Anti-gpc3 antibody

ABSTRACT

An object of the present invention is to provide: an anti-GPC3 antibody that recognizes an epitope different from that for existing antibodies (e.g., GC33 and GC199) and can specifically bind, even in the form of single chain antibody, to GPC3 localized on a cell membrane; CAR comprising the anti-GPC3 single chain antibody; an immunocompetent cell expressing the CAR; a gene of the anti-GPC3 antibody or a gene of the CAR; a vector comprising the anti-GPC3 antibody gene or the CAR gene; a host cell in which the vector has been introduced; a method for specifically detecting GPC3; and a kit for specifically detecting GPC3. An antibody comprising particular heavy chain CDR1 to CDR3 and particular light chain CDR1 to CDR3 defined in claim  1 , and specifically binding to a human-derived GPC3 polypeptide specifically binds to GPC3 localized on a cell membrane. CAR-immunocompetent cells prepared on the basis of CAR comprising such single chain antibody are useful for cancer immunotherapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No. 16/472,356, which is the U.S. National Stage of PCT/JP2018/000257, filed Jan. 10, 2018, which claims priority to JP 2017-001732, filed Jan. 10, 2017.

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-WEB and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 14, 2019, is named sequence.txt and is 207903 bytes.

TECHNICAL FIELD

The present invention relates to: an antibody specifically binding to GPC3 (glypican-3) (anti-GPC3 antibody); a chimeric antigen receptor (hereinafter, also referred to as “CAR”) comprising anti-GPC3 single chain antibody, a transmembrane region fused with a carboxyl (C) terminus of the anti-GPC3 single chain antibody, and an immunocompetent cell activation signal transduction region fused with a C terminus of the transmembrane region; an immunocompetent cell expressing the CAR; an anti-GPC3 antibody gene or a CAR gene; a vector comprising the anti-GPC3 antibody gene or the CAR gene; a host cell in which the vector has been introduced; a method for detecting GPC3; and a kit for detecting GPC3.

BACKGROUND ART

Glypican-3 (GPC3) is an extracellular matrix protein that is expressed in embryonic tissues, particularly, the liver or the kidney, and associated with organogenesis. The expression of GPC3 is not observed in human adult tissues except for placenta, but is observed in tissues of various cancers such as hepatocellular carcinoma, melanoma, ovarian clear cell adenocarcinoma, and lung squamous cell carcinoma. Thus, GPC3 is a protein that is expressed in embryonic tissues, as in proteins such as α-fetoprotein (AFP) and carcinoembryonic antigen (CEA), and is therefore classified into embryonal carcinoma antigens. Specifically, GPC3 is useful as a target molecule of cancer treatment, a tumor marker and a diagnostic marker, because its feature is that the protein is not expressed in normal tissue cells, but is specifically expressed in cancer cells.

GPC3 is a member of the proteoglycan family that functions as extracellular matrix in cell adhesion in organogenesis or as a receptor of a cell growth factor. A GPI (glycosylphosphatidylinositol) anchor is added to serine at position 560 located on the carboxyl (C)-terminal side of GPC3. The GPI anchor plays a role in localizing GPC3 on cell surface through covalent binding to cell membrane lipid. Also, serine at position 495 and serine at position 509 of GPC3 are modified with a heparan sulfate chain (HS chain). The HS chain is known to regulate a plurality of growth signal transduction pathways such as Wnt signal, FGF signal, and BMP signal transduction pathways. A growth signal transduction pathway involved is known to differ among the types of cancers. For example, in hepatocellular carcinoma (HCC), cells grow by the stimulation of the Wnt signal pathway. A common feature of the glypican family is the number of cysteine as abundant as 16 in an extracellular region, and these cysteine residues are considered to contribute to the stable formation of a conformation by forming a plurality of intramolecular disulfide bonds. The possibility has been reported that GPC3 on cell membrane surface is cleaved between arginine (R) at position 358 and serine (S) at position 359 (R358/S359) by furin convertase. However, since an amino (N)-terminal subunit of GPC3 is cross-linked through intramolecular disulfide bonds, GPC3, even when cleaved into two subunits, an N-terminal subunit and a C-terminal subunit, by furin convertase may probably retain its full-length structure without dissociating these subunits. The structure of soluble GPC3 remains a controversial subject. Thus, there are many unclear points as to the conformation of GPC3 localized on a cell membrane, also including the structures of isoforms of GPC3.

GPC3 on a cell membrane has a complicated structure. Therefore, for preparing an antibody against GPC3, it has been considered desirable that the simplest structural region is an epitope. A representative existing anti-GPC3 antibody includes a monoclonal antibody 1G12 which is distributed by BioMosaics, Inc. This antibody is an antibody obtained by immunizing Balb/c mice with an antigen (C-terminal 70-residue polypeptide of GPC3) designed so as to circumvent the complicated structure or localization of GPC3, to prepare hybridomas, and screening the hybridomas using the antigen. Antibodies GC33 and GC199 developed by a Japanese pharmaceutical manufacturer are also monoclonal antibodies established on the basis of the same concept as above and are antibodies obtained with the C-terminal partial fragment of GPC3 as an antigen (patent document 1).

PRIOR ART DOCUMENT Patent Document

Patent document 1: Japanese Patent No. 4011100

SUMMARY OF THE INVENTION Object to be Solved by the Invention

An object of the present invention is to provide: an anti-GPC3 antibody that recognizes an epitope different from that for existing antibodies (e.g., GC33 and GC199) and can specifically bind, even in the form of single chain antibody, to GPC3 localized on a cell membrane; CAR comprising the anti-GPC3 single chain antibody; an immunocompetent cell expressing the CAR; a gene of the anti-GPC3 antibody or a gene of the CAR; a vector comprising the anti-GPC3 antibody gene or the CAR gene; a host cell in which the vector has been introduced; a method for specifically detecting GPC3; and a kit for specifically detecting GPC3.

Means to Solve the Object

The present inventors are continuing diligent studies to attain the object. In the course of the studies, the present inventors have prepared a novel anti-GPC3 antibody by a phage display method which is an approach different from conventional monoclonal antibody preparation methods involving establishing hybridomas. Specifically, an immune library of antibody genes was synthesized using B cells derived from mice immunized with full-length human GPC3, and the genes were reconstituted into a single chain antibody (scFv) library, which was then incorporated into a phage display and expressed on phage surface, followed by biopanning using recombinant full-length human GPC3 and the GPC3-expressing cell line, and further, if necessary, a competitor C-terminal polypeptide of GPC3 serving as an epitope for the existing antibodies, to prepare an anti-GPC3 antibody. The prepared anti-GPC3 antibody has also been confirmed to be useful for cancer immunotherapy using T cells expressing a chimeric antigen receptor (CAR) (hereinafter, also referred to as “CAR-T cells”). The present invention has been completed on the basis of these findings.

Specifically, the present invention is as follows.

[1] An antibody specifically binding to a human GPC3 (glypican-3)-derived polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 155 (hereinafter, also referred to as the “present antibody”), wherein the antibody

(1-1) comprises a heavy chain complementarity determining region (CDR) 1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3, and

a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6; or

(2-1) comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 12, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 13, and

a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 14, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 15, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 16; or

(3-1) comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 23, and

a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 24, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 25, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 26; or

(4-1) comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 32, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 33, and

a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 36; or

(5-1) comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 42, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 43, and

a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 44, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 45, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 46; or

(6-1) comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 51, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 52, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 53, and

a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 54, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 55, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 56; or

(7-1) comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 61, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 62, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 63, and

a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 64, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 65, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 66; or

(8-1) comprises heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 71, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 72, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 73, and

a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 74, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 75, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 76; or

(9-1) comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 81, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 82, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 83, and

a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 84, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 85, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 86; or

(10-1) comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 91, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 92, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 93, and

a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 94, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 95, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 96; or

(11-1) comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 101, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 102, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 103, and

a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 104, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 105, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 106.

[2] The antibody according to [1], wherein the antibody

(1-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 7, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 8; or

(2-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 17, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 18; or

(3-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 27, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 28; or

(4-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 37, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 38; or

(5-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 47, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 48; or

(6-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 57, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 58; or

(7-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 67, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 68; or

(8-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 77, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 78; or

(9-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 87, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 88; or

(10-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 97, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 98; or

(11-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 107, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 108.

[3] The antibody according to [1] or [2], wherein the antibody is single chain antibody. [4] The antibody according to [3], wherein the single chain antibody

(1-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 165; or

(2-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 166; or

(3-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 167; or

(4-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 168; or

(5-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 169; or

(6-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 170; or

(7-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 171; or

(8-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 172; or

(9-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 173; or

(10-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 174; or

(11-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 175.

[5] The antibody according to [3], wherein the single chain antibody

(1-3′-1) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 178; or

(1-3′-2) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 179; or

(1-3′-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 180; or

(2-3′-1) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 181; or

(2-3′-2) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 182; or

(2-3′-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 183; or

(2-3′-4) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 184.

[6] The antibody according to [1] or [2], wherein the antibody

(1-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 9, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 10; or

(2-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 19, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 20; or

(3-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 29, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 30; or

(4-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 39, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 40; or

(5-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 49, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 50; or

(6-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 59, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 60; or

(7-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 69, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 70; or

(8-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 79, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 80; or

(9-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 89, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 90; or

(10-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 99, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 100; or

(11-4) comprises a heavy chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 109, and a light chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 110.

[7] CAR comprising the antibody according to any one of [3] to [5] (hereinafter, also referred to as the “present single chain antibody”), a transmembrane region fused with a carboxyl terminus of the present single chain antibody, and an immunocompetent cell activation signal transduction region fused with a carboxyl terminus of the transmembrane region (hereinafter, also referred to as the “present CAR”). [8] The CAR according to [7], comprising the amino acid sequence represented by any of SEQ ID NOs: 185 to 187. [9] An immunocompetent cell expressing the CAR according to [7] or [8] (hereinafter, also referred to as the “present immunocompetent cell”). [10] The immunocompetent cell according to [9], further expressing interleukin 7 (IL-7) and chemokine ligand 19 (CCL19). [11] An antibody gene encoding the antibody according to any one of [1] to [6] (hereinafter, also referred to as the “present antibody gene”), or a CAR gene encoding the CAR according to [7] or [8] (hereinafter, also referred to as the “present CAR gene”). [12] An antibody gene encoding the antibody according to any one of [1] to [4] and [6]. [13] A vector comprising a promoter, and the antibody gene according to [11] or the CAR gene encoding the CAR according to [11] operably linked downstream of the promoter (hereinafter, also referred to as the “present vector”). [14] A vector comprising a promoter, and the antibody gene according to [12] operably linked downstream of the promoter. [15] A host cell in which the vector according to [13] or [14] has been introduced (hereinafter, also referred to as the “present host cell”). [16] A method for detecting GPC3 (glypican-3), comprising the step of detecting GPC3 using the antibody according to any one of [1] to [6] (hereinafter, also referred to as the “present detection method”). [17] A kit for the detection of GPC3 (glypican-3), comprising the antibody according to any one of [1] to [6], or a labeled form thereof (hereinafter, also referred to as the “present kit for detection”).

Examples of other embodiments of the present invention can include the present antibody for use in the detection of GPC3, and a method for producing the present antibody, comprising the steps of: immunizing nonhuman animals (e.g., mice and rats) with full-length human GPC3 consisting of the amino acid sequence represented by SEQ ID NO: 157; synthesizing cDNA by reverse transcription reaction from total RNA of B cells derived from the immunized nonhuman animals, and amplifying antibody genes to prepare an antibody gene library; and constructing a scFv phage library from the antibody gene library, and infecting E. coli with the library so that cells express scFv, followed by biopanning using the full-length human GPC3 and the GPC3-expressing cell line, and further, if necessary, a competitor C-terminal polypeptide of GPC3 (human-derived GPC3 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 156).

Effect of the Invention

The present antibody is an antibody specifically binding to GPC3 localized on a cell membrane not only in the form of IgG but in the form of scFv. CAR-T cells using the present antibody as scFv in CAR have excellent cytotoxic activity and the ability to produce IFN-γ. Hence, the present antibody is useful for cancer immunotherapy.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing each round (step) of biopanning consisting of 5 types of series (A to E series). A series involves performing 3 rounds of biopanning with recombinant GPC3 immobilized on magnetic beads as a bait, and performing biopanning in rounds 4 and 5 with a GPC3-expressing cell line as a bait (round 5 was carried out only for 1413 #3). In rounds 1 to 4, existing anti-GPC3 antibodies (GC33 and GC199) were added as competitive antibodies. B series involves performing biopanning with GPC3-expressing cells as a bait in the presence of the competitive antibodies after round 2 of A series. E series involves performing biopanning with recombinant GPC3 immobilized on magnetic beads as a bait under conditions of no competitive antibody after round 3 of A series. In C series, 4 rounds in total of biopanning with a GPC3-expressing cell line as a bait in 2 rounds and recombinant GPC3 immobilized on magnetic beads as a bait in 2 rounds were performed in the absence of the competitive antibodies. D series involves performing the same biopanning as that of A series in the absence of the competitive antibodies.

FIG. 2 is a diagram showing results of performing flow cytometry (FCM) using 18 types of anti-GPC3 scFv clones (TF1413-02d023, 02d028, 02d030, 02d039, 02e003, 02e004, 02e014, 02e030, 02e040, 03e001, 03e004, 03e005, 03e015, 03e016, 03e019, 03e027, 03e034, and 03e045) and existing anti-GPC3 antibodies (GC33 and GC199), and 3 types of cell lines (GPC3 N-terminal fragment-expressing cell line, GPC3 C-terminal fragment-expressing cell line, and GPC3 [full-length]-expressing cell line). The numeric values in the diagram are indicated by relative values when the fluorescence intensity of a cell line expressing no GPC3 (SK-Hep-1 cell line) was defined as 1 in FCM.

FIG. 3 is a diagram showing results of performing FCM using IgG antibodies prepared from 11 types of scFv clones (TF1413-02d028, 02d039, 02e004, 02e014, 02e030, 02e040, 03e001, 03e004, 03e005, 03e015, and 03e034) and existing anti-GPC3 antibodies (GC33 and GC199), and 3 types of cell lines (GPC3 N-terminal fragment-expressing cell line, GPC3 C-terminal fragment-expressing cell line, and GPC3 [full-length]-expressing cell line).

FIG. 4 is a diagram showing results of performing FACS (fluorescence activated cell sorting) using a GPC3-expressing cell line treated with 3 types of methods (EDTA, trypsin, and “EDTA+collagenase”), 3 types of antibody combinations (anti-mouse IgG antibody labeled with APC [hereinafter, also referred to as “APC ant-mouse IgG antibody”], and a combination of the APC anti-mouse IgG antibody and a scFv clone [TF1413-02d028] antibody).

FIG. 5 is a diagram showing results of analyzing GPC3 CAR-T cells (T cells expressing CAR of scFv recognizing GPC3) derived from 5 types of scFv clones (TF1413-02d028, TF1413-02d039, TF1413-02e014, TF1413-02e030, and TF1413-03e005) for cytotoxic activity against a Sk-HEP-1 GPC3 cell line. In each graph, the right peak depicts CD45-positive cells (GPC3 CAR-T cells), and the left peak depicts CD45-negative cells (residual cancer cells [Sk-HEP-1 GPC3 cells]). The ordinate of each graph depicts the number of cells. The numeric value in each graph depicts the ratio (%) of the number of CD45-positive cells to the total number of cells (CD45-positive cells and CD45-negative cells). T cells expressing no GPC3 CAR (“Non infection” in the diagram) were used as a control.

FIGS. 6A and 6B are graphs showing the ratio of CD45-negative cells in FIG. 5 (FIG. 6A) and the number of CD45-negative cells (FIG. 6B). In a pair of bar graphs, the left bar graph depicts “mock” (Sk-HEP-1 mock cell line), and the right bar graph depicts “GPC3” (Sk-HEP-1 GPC3 cell line).

FIG. 7 is a diagram showing results of analyzing GPC3 CAR-T cells derived from 5 types of scFv clones (TF1413-02d028, TF1413-02d039, TF1413-02e014, TF1413-02e030, and TF1413-03e005) for the ability to produce IFN-γ against a Sk-HEP-1 GPC3 cell line. T cells expressing no GPC3 CAR (“Non infection” in the diagram) were used as a control.

MODE OF CARRYING OUT THE INVENTION

The present antibody is an antibody comprising the heavy (H) chain and light (L) chain CDR1 to CDR3 described above in any of (1-1) to (11-1), and specifically binding to, as an epitope, at least a portion (usually within the range of 3 to 30 amino acid residues, preferably 4 to 20 amino acid residues, more preferably 5 to 15 amino acid residues) of a human-derived GPC3 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 155 (amino [N]-terminal polypeptide consisting of amino acid residues 32 to 471 [exons 1 to 7] of human-derived full-length GPC3 consisting of the amino acid sequence represented by SEQ ID NO: 157). This antibody specifically binds not only in the form of IgG but in the form of scFv to GPC3 localized on a cell membrane, and usually comprises a H chain variable region comprising the H chain CDR1 to CDR3 described above in any of (1-1) to (11-1), and a L chain variable region comprising the L chain CDR1 to CDR3 described above in any of (1-1) to (11-1). In this context, the phrase “specifically binding” means that the antibody recognizes and binds to the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 155 through a recognition mechanism with high antigen-antibody specificity. Thus, the present antibody does not specifically bind to a human-derived GPC3 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 156 (carboxyl [C]-terminal polypeptide consisting of amino acid residues 472 to 580 [exons 8 and 9] of human-derived full-length GPC3 consisting of the amino acid sequence represented by SEQ ID NO: 157).

The present antibody is not particularly limited by its origin, type, class, morphology, etc. The present antibody includes, for example: a human-derived antibody; an antibody derived from a nonhuman animal such as a mouse or a rat; a polyclonal antibody, an oligoclonal antibody (mixture of several to several tens of antibodies), and a monoclonal antibody; and a chimeric antibody or a humanized antibody in which a partial region (e.g., constant regions) of an antibody has been substituted by a region derived from a different organism species, an antibody fragment such as a F(ab′)₂ antibody fragment obtained by digesting a monoclonal antibody with pepsin, a Fab′ antibody fragment obtained by reducing a F(ab′)₂ antibody fragment, and Fab obtained by digesting a monoclonal antibody with papain, and a recombinant antibody such as scFv containing an antibody heavy (H) chain variable region and an antibody light (H) chain variable region linked through amino acid cross-links. In the case of using the present antibody as CAR, scFv is preferred.

The present antibody is preferably in a separated form. In this context, the term “separated” means that the antibody is present in a state different from the state where the antibody is originally present in such a way that the antibody is taken out of an environment originally involving the antibody or expressed in an environment different from the environment originally involving the antibody by an artificial operation. Specifically, the “separated antibody” does not include an antibody that is derived from a certain individual and is in a state contained in the body of the individual without an external operation (artificial operation) or in a tissue or a body fluid (blood, plasma, serum, etc.) derived from the body. The present antibody is preferably an antibody prepared by an artificial operation (e.g., the recombinant antibody described above). Such an “antibody derived from a cell prepared by an artificial operation or an antibody produced from the cell” does not include an antibody that is not subjected to an artificial operation, for example, an antibody produced from a naturally occurring B cell.

In the present antibody, a framework region (FR) is usually linked to the N terminus and/or C terminus of each of H chain and L chain CDR1 to CDR3 regions. Among such FRs, examples of the H chain FRs can include H chain FR1 linked to the N terminus of H chain CDR1, H chain FR2 linked to the C terminus of H chain CDR1 (N terminus of H chain CDR2), H chain FR3 linked to the C terminus of H chain CDR2 (N terminus of H chain CDR3), and H chain FR4 linked to the C terminus of H chain CDR3. Among the FRs, examples of the L chain FRs can include L chain FR1 linked to the N terminus of L chain CDR1, L chain FR2 linked to the C terminus of L chain CDR1 (N terminus of L chain CDR2), L chain FR3 linked to the C terminus of L chain CDR2 (N terminus of L chain CDR3), and L chain FR4 linked to the C terminus of L chain CDR3.

Examples of the H chain FR1 can specifically include: (1-HFR1) a polypeptide consisting of amino acid residues 1 to 30 of the amino acid sequence represented by SEQ ID NO: 7, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (2-HFR1) a polypeptide consisting of amino acid residues 1 to 30 of the amino acid sequence represented by SEQ ID NO: 17, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (3-HFR1) a polypeptide consisting of amino acid residues 1 to 30 of the amino acid sequence represented by SEQ ID NO: 27, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (4-HFR1) a polypeptide consisting of amino acid residues 1 to 30 of the amino acid sequence represented by SEQ ID NO: 37, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (5-HFR1) a polypeptide consisting of amino acid residues 1 to 30 of the amino acid sequence represented by SEQ ID NO: 47, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (6-HFR1) a polypeptide consisting of amino acid residues 1 to 30 of the amino acid sequence represented by SEQ ID NO: 57, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (7-HFR1) a polypeptide consisting of amino acid residues 1 to 30 of the amino acid sequence represented by SEQ ID NO: 67, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (8-HFR1) a polypeptide consisting of amino acid residues 1 to 30 of the amino acid sequence represented by SEQ ID NO: 77, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (9-HFR1) a polypeptide consisting of amino acid residues 1 to 30 of the amino acid sequence represented by SEQ ID NO: 87, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (10-HFR1) a polypeptide consisting of amino acid residues 1 to 30 of the amino acid sequence represented by SEQ ID NO: 97, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; and (11-HFR1) a polypeptide consisting of amino acid residues 1 to 30 of the amino acid sequence represented by SEQ ID NO: 107, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide.

Examples of the H chain FR2 can specifically include: (1-HFR2) a polypeptide consisting of amino acid residues 36 to 49 of the amino acid sequence represented by SEQ ID NO: 7, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (2-HFR2) a polypeptide consisting of amino acid residues 36 to 49 of the amino acid sequence represented by SEQ ID NO: 17, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (3-HFR2) a polypeptide consisting of amino acid residues 36 to 49 of the amino acid sequence represented by SEQ ID NO: 27, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (4-HFR2) a polypeptide consisting of amino acid residues 36 to 49 of the amino acid sequence represented by SEQ ID NO: 37, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (5-HFR2) a polypeptide consisting of amino acid residues 36 to 49 of the amino acid sequence represented by SEQ ID NO: 47, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (6-HFR2) a polypeptide consisting of amino acid residues 36 to 49 of the amino acid sequence represented by SEQ ID NO: 57, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (7-HFR2) a polypeptide consisting of amino acid residues 36 to 49 of the amino acid sequence represented by SEQ ID NO: 67, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (8-HFR2) a polypeptide consisting of amino acid residues 36 to 49 of the amino acid sequence represented by SEQ ID NO: 77, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (9-HFR2) a polypeptide consisting of amino acid residues 36 to 49 of the amino acid sequence represented by SEQ ID NO: 87, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (10-HFR2) a polypeptide consisting of amino acid residues 36 to 49 of the amino acid sequence represented by SEQ ID NO: 97, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; and (11-HFR2) a polypeptide consisting of amino acid residues 36 to 49 of the amino acid sequence represented by SEQ ID NO: 107, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide.

Examples of the H chain FR3 can specifically include: (1-HFR3) a polypeptide consisting of amino acid residues 67 to 98 of the amino acid sequence represented by SEQ ID NO: 7, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (2-HFR3) a polypeptide consisting of amino acid residues 67 to 98 of the amino acid sequence represented by SEQ ID NO: 17, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (3-HFR3) a polypeptide consisting of amino acid residues 67 to 98 of the amino acid sequence represented by SEQ ID NO: 27, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (4-HFR3) a polypeptide consisting of amino acid residues 67 to 99 of the amino acid sequence represented by SEQ ID NO: 37, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (5-HFR3) a polypeptide consisting of amino acid residues 67 to 99 of the amino acid sequence represented by SEQ ID NO: 47, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (6-HFR3) a polypeptide consisting of amino acid residues 67 to 98 of the amino acid sequence represented by SEQ ID NO: 57, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (7-HFR3) a polypeptide consisting of amino acid residues 67 to 98 of the amino acid sequence represented by SEQ ID NO: 67, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (8-HFR3) a polypeptide consisting of amino acid residues 67 to 98 of the amino acid sequence represented by SEQ ID NO: 77, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (9-HFR3) a polypeptide consisting of amino acid residues 67 to 99 of the amino acid sequence represented by SEQ ID NO: 87, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (10-HFR3) a polypeptide consisting of amino acid residues 67 to 98 of the amino acid sequence represented by SEQ ID NO: 97, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; and (11-HFR3) a polypeptide consisting of amino acid residues 67 to 98 of the amino acid sequence represented by SEQ ID NO: 107, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide.

Examples of the H chain FR4 can specifically include: (1-HFR4) a polypeptide consisting of amino acid residues 109 to 118 of the amino acid sequence represented by SEQ ID NO: 7, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (2-HFR4) a polypeptide consisting of amino acid residues 108 to 117 of the amino acid sequence represented by SEQ ID NO: 17, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (3-HFR4) a polypeptide consisting of amino acid residues 106 to 115 of the amino acid sequence represented by SEQ ID NO: 27, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (4-HFR4) a polypeptide consisting of amino acid residues 111 to 120 of the amino acid sequence represented by SEQ ID NO: 37, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (5-HFR4) a polypeptide consisting of amino acid residues 108 to 117 of the amino acid sequence represented by SEQ ID NO: 47, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (6-HFR4) a polypeptide consisting of amino acid residues 107 to 116 of the amino acid sequence represented by SEQ ID NO: 57, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (7-HFR4) a polypeptide consisting of amino acid residues 106 to 115 of the amino acid sequence represented by SEQ ID NO: 67, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (8-HFR4) a polypeptide consisting of amino acid residues 106 to 115 of the amino acid sequence represented by SEQ ID NO: 77, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (9-HFR4) a polypeptide consisting of amino acid residues 111 to 120 of the amino acid sequence represented by SEQ ID NO: 87, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (10-HFR4) a polypeptide consisting of amino acid residues 110 to 119 of the amino acid sequence represented by SEQ ID NO: 97, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; and (11-HFR4) a polypeptide consisting of amino acid residues 109 to 118 of the amino acid sequence represented by SEQ ID NO: 107, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide.

Examples of the L chain FR1 can specifically include: (1-LFR1) a polypeptide consisting of amino acid residues 1 to 23 of the amino acid sequence represented by SEQ ID NO: 8, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (2-LFR1) a polypeptide consisting of amino acid residues 1 to 23 of the amino acid sequence represented by SEQ ID NO: 18, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (3-LFR1) a polypeptide consisting of amino acid residues 1 to 23 of the amino acid sequence represented by SEQ ID NO: 28, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (4-LFR1) a polypeptide consisting of amino acid residues 1 to 23 of the amino acid sequence represented by SEQ ID NO: 38, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (5-LFR1) a polypeptide consisting of amino acid residues 1 to 23 of the amino acid sequence represented by SEQ ID NO: 48, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (6-LFR1) a polypeptide consisting of amino acid residues 1 to 23 of the amino acid sequence represented by SEQ ID NO: 58, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (7-LFR1) a polypeptide consisting of amino acid residues 1 to 23 of the amino acid sequence represented by SEQ ID NO: 68, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (8-LFR1) a polypeptide consisting of amino acid residues 1 to 23 of the amino acid sequence represented by SEQ ID NO: 78, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (9-LFR1) a polypeptide consisting of amino acid residues 1 to 23 of the amino acid sequence represented by SEQ ID NO: 88, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (10-LFR1) a polypeptide consisting of amino acid residues 1 to 23 of the amino acid sequence represented by SEQ ID NO: 98, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; and (11-LFR1) a polypeptide consisting of amino acid residues 1 to 23 of the amino acid sequence represented by SEQ ID NO: 108, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide.

Examples of the L chain FR2 can specifically include: (1-LFR2) a polypeptide consisting of amino acid residues 35 to 49 of the amino acid sequence represented by SEQ ID NO: 8, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (2-LFR2) a polypeptide consisting of amino acid residues 40 to 54 of the amino acid sequence represented by SEQ ID NO: 18, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (3-LFR2) a polypeptide consisting of amino acid residues 35 to 49 of the amino acid sequence represented by SEQ ID NO: 28, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (4-LFR2) a polypeptide consisting of amino acid residues 35 to 49 of the amino acid sequence represented by SEQ ID NO: 38, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (5-LFR2) a polypeptide consisting of amino acid residues 41 to 55 of the amino acid sequence represented by SEQ ID NO: 48, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (6-LFR2) a polypeptide consisting of amino acid residues 35 to 49 of the amino acid sequence represented by SEQ ID NO: 58, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (7-LFR2) a polypeptide consisting of amino acid residues 35 to 49 of the amino acid sequence represented by SEQ ID NO: 68, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (8-LFR2) a polypeptide consisting of amino acid residues 35 to 49 of the amino acid sequence represented by SEQ ID NO: 78, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (9-LFR2) a polypeptide consisting of amino acid residues 35 to 49 of the amino acid sequence represented by SEQ ID NO: 88, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (10-LFR2) a polypeptide consisting of amino acid residues 35 to 49 of the amino acid sequence represented by SEQ ID NO: 98, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; and (11-LFR2) a polypeptide consisting of amino acid residues 35 to 49 of the amino acid sequence represented by SEQ ID NO: 108, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide.

Examples of the L chain FR3 can specifically include: (1-LFR3) a polypeptide consisting of amino acid residues 57 to 88 of the amino acid sequence represented by SEQ ID NO: 8, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (2-LFR3) a polypeptide consisting of amino acid residues 62 to 93 of the amino acid sequence represented by SEQ ID NO: 18, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (3-LFR3) a polypeptide consisting of amino acid residues 57 to 88 of the amino acid sequence represented by SEQ ID NO: 28, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (4-LFR3) a polypeptide consisting of amino acid residues 57 to 88 of the amino acid sequence represented by SEQ ID NO: 38, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (5-LFR3) a polypeptide consisting of amino acid residues 63 to 94 of the amino acid sequence represented by SEQ ID NO: 48, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (6-LFR3) a polypeptide consisting of amino acid residues 57 to 88 of the amino acid sequence represented by SEQ ID NO: 58, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (7-LFR3) a polypeptide consisting of amino acid residues 57 to 88 of the amino acid sequence represented by SEQ ID NO: 68, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (8-LFR3) a polypeptide consisting of amino acid residues 57 to 88 of the amino acid sequence represented by SEQ ID NO: 78, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (9-LFR3) a polypeptide consisting of amino acid residues 57 to 88 of the amino acid sequence represented by SEQ ID NO: 88, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (10-LFR3) a polypeptide consisting of amino acid residues 57 to 88 of the amino acid sequence represented by SEQ ID NO: 98, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; and (11-LFR3) a polypeptide consisting of amino acid residues 57 to 88 of the amino acid sequence represented by SEQ ID NO: 108, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide.

Examples of the L chain FR4 can specifically include: (1-LFR4) a polypeptide consisting of amino acid residues 98 to 108 of the amino acid sequence represented by SEQ ID NO: 8, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (2-LFR4) a polypeptide consisting of amino acid residues 103 to 113 of the amino acid sequence represented by SEQ ID NO: 18, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (3-LFR4) a polypeptide consisting of amino acid residues 97 to 107 of the amino acid sequence represented by SEQ ID NO: 28, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (4-LFR4) a polypeptide consisting of amino acid residues 98 to 108 of the amino acid sequence represented by SEQ ID NO: 38, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (5-LFR4) a polypeptide consisting of amino acid residues 104 to 114 of the amino acid sequence represented by SEQ ID NO: 48, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (6-LFR4) a polypeptide consisting of amino acid residues 98 to 108 of the amino acid sequence represented by SEQ ID NO: 58, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (7-LFR4) a polypeptide consisting of amino acid residues 98 to 108 of the amino acid sequence represented by SEQ ID NO: 68, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (8-LFR4) a polypeptide consisting of amino acid residues 98 to 108 of the amino acid sequence represented by SEQ ID NO: 78, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (9-LFR4) a polypeptide consisting of amino acid residues 98 to 108 of the amino acid sequence represented by SEQ ID NO: 88, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; (10-LFR4) a polypeptide consisting of amino acid residues 98 to 108 of the amino acid sequence represented by SEQ ID NO: 98, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide; and (11-LFR4) a polypeptide consisting of amino acid residues 98 to 108 of the amino acid sequence represented by SEQ ID NO: 108, or a polypeptide consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence of the polypeptide.

The FRs of the present antibody are preferably FRs of a known human antibody. Examples of such “FRs of a known human antibody” can include FRs of a human antibody registered in a sequence database known in the art such as GenBank, and FRs selected from a common sequence (human most homologous consensus sequence; Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991) derived from each subgroup of a human antibody.

The H chain CDR1 in the present antibody usually resides at positions H31 to H35 based on Kabat numbering (see the document “Kabat, E. A. et al., (1991) NIH Publication No. 91-3242, sequences of proteins of immunological interest”). The H chain CDR2 in the present antibody usually resides at positions H50 to H52, H52A, and H53 to H65 based on Kabat numbering. The H chain CDR3 in the present antibody usually resides at positions H95 to H100, H100A, H100B, H101, and H102 based on Kabat numbering. The L chain CDR1 in the present antibody usually resides at positions L24 to L34 based on Kabat numbering. The L chain CDR2 in the present antibody usually resides at positions L50 to L56 based on Kabat numbering. The L chain CDR3 in the present antibody usually resides at positions L89 to L97 based on Kabat numbering.

Examples of the antibody comprising the H chain and L chain CDR1 to CDR3 described above in (1-1) as the present antibody can include an antibody comprising the H chain and L chain variable (V) regions described above in (1-2) and can specifically include: the single chain antibody described above in (1-3); the single chain antibody described above in (1-3′-1), the single chain antibody described above in (1-3′-2), and the single chain antibody described above in (1-3′-3); and an antibody comprising the H chain and the L chain described above in (1-4). Examples of the antibody comprising the H chain and L chain CDR1 to CDR3 described above in (2-1) can include an antibody comprising the H chain and L chain V regions described above in (2-2) and can specifically include: the single chain antibody described above in (2-3); the single chain antibody described above in (2-3′-1), the single chain antibody described above in (2-3′-2), the single chain antibody described above in (2-3′-3), and the single chain antibody described above in (2-3′-4); and an antibody comprising the H chain and the L chain described above in (2-4). Examples of the antibody comprising the H chain and L chain CDR1 to CDR3 described above in (3-1) can include an antibody comprising the H chain and L chain V regions described above in (3-2) and can specifically include: the single chain antibody described above in (3-3); and an antibody comprising the H chain and the L chain described above in (3-4). Examples of the antibody comprising the H chain and L chain CDR1 to CDR3 described above in (4-1) can include an antibody comprising the H chain and L chain V regions described above in (4-2) and can specifically include: the single chain antibody described above in (4-3); and an antibody comprising the H chain and the L chain described above in (4-4). Examples of the antibody comprising the H chain and L chain CDR1 to CDR3 described above in (5-1) can include an antibody comprising the H chain and L chain V regions described above in (5-2) and can specifically include: the single chain antibody described above in (5-3); and an antibody comprising the H chain and the L chain described above in (5-4). Examples of the antibody comprising the H chain and L chain CDR1 to CDR3 described above in (6-1) can include an antibody comprising the H chain and L chain V regions described above in (6-2) and can specifically include: the single chain antibody described above in (6-3); and an antibody comprising the H chain and the L chain described above in (6-4). Examples of the antibody comprising the H chain and L chain CDR1 to CDR3 described above in (7-1) can include an antibody comprising the H chain and L chain V regions described above in (7-2) and can specifically include: the single chain antibody described above in (7-3); and an antibody comprising the H chain and the L chain described above in (7-4). Examples of the antibody comprising the H chain and L chain CDR1 to CDR3 described above in (8-1) can include an antibody comprising the H chain and L chain V regions described above in (8-2) and can specifically include: the single chain antibody described above in (8-3); and an antibody comprising the H chain and the L chain described above in (8-4). Examples of the antibody comprising the H chain and L chain CDR1 to CDR3 described above in (9-1) can include an antibody comprising the H chain and L chain V regions described above in (9-2) and can specifically include: the single chain antibody described above in (9-3); and an antibody comprising the H chain and the L chain described above in (9-4). Examples of the antibody comprising the H chain and L chain CDR1 to CDR3 described above in (10-1) can include an antibody comprising the H chain and L chain V regions described above in (10-2) and can specifically include: the single chain antibody described above in (10-3); and an antibody comprising the H chain and the L chain described above in (10-4). Examples of the antibody comprising the H chain and L chain CDR1 to CDR3 described above in (11-1) can include an antibody comprising the H chain and L chain V regions described above in (11-2) and can specifically include: the single chain antibody described above in (11-3); and an antibody comprising the H chain and the L chain described above in (11-4). The heavy chain variable region and the light chain variable region in the single chain antibody are usually bound via a peptide linker.

The present CAR can comprise the present single chain antibody, a transmembrane region fused with the C terminus of the present single chain antibody, and an immunocompetent cell activation signal transduction region fused with the C terminus of the transmembrane region. In this context, the fusion between the present single chain antibody and the transmembrane region, or between the transmembrane region and the immunocompetent cell activation signal transduction region may be mediated by a peptide linker or an IgG4 hinge region.

Examples of the length of the peptide linker in the present antibody can include 1 to 100 amino acid residues, preferably 10 to 50 amino acid residues. Examples of the peptide linker in the present antibody can specifically include a consecutive linkage of 3 amino acid sequences each consisting of 1 to 4 glycine residues and 1 serine residue.

The transmembrane region can be any peptide that can penetrate a cell membrane. Examples thereof can include a transmembrane region derived from CD8, a T cell receptor α or β chain, CD3ζ, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, EGFR (epidermal growth factor receptor), or GITR and can specifically include a human CD8 transmembrane region consisting of amino acid residues 1 to 83 of the amino acid sequence represented by SEQ ID NO: 185. Alternatively, the transmembrane region may be derived from a peptide that can penetrate cell membrane by the truncation of C-terminal 1 to 10 amino acid residues, preferably 6 or 7 amino acid residues. Examples thereof can include engineered form 1 of the human CD8 transmembrane region consisting of amino acid residues 1 to 77 of the amino acid sequence represented by SEQ ID NO: 186, and engineered form 2 of the human CD8 transmembrane region consisting of amino acid residues 1 to 76 of the amino acid sequence represented by SEQ ID NO: 187.

The immunocompetent cell activation signal transduction region can be any region capable of transducing a signal into immunocompetent cells upon binding of the present single chain antibody to human GPC3. The immunocompetent cell activation signal transduction region preferably comprises at least one or more members selected from polypeptides of the intracellular regions of CD28, 4-1BB (CD137), GITR, CD27, OX40, HVEM, CD3ζ, and Fc receptor-associated γ chain, and more preferably comprises three polypeptides of the intracellular regions of CD28, 4-1BB, and CD3ζ. Examples of such a polypeptide of the intracellular region of CD28 can specifically include a polypeptide of the intracellular region of human CD28 consisting of amino acid residues 85 to 124 of the amino acid sequence represented by SEQ ID NO: 185. Examples of the “polypeptide of the intracellular region of 4-1BB” can specifically include a polypeptide of the intracellular region of human 4-1BB consisting of amino acid residues 125 to 170 of the amino acid sequence represented by SEQ ID NO: 185. Examples of the polypeptide of the intracellular region of CD3ζ can specifically include a polypeptide of the intracellular region of human CD3ζ consisting of amino acid residues 172 to 283 of the amino acid sequence represented by SEQ ID NO: 185.

Arginine (Arg) at position 84 of the amino acid sequence represented by SEQ ID NO: 185, arginine at position 78 of the amino acid sequence represented by SEQ ID NO: 186, and arginine at position 77 of the amino acid sequence represented by SEQ ID NO: 187 are a common sequence between the polypeptide of the transmembrane region derived from human CD8 and the polypeptide of the intracellular region of human CD28. Leucine (Leu) at position 171 of the amino acid sequence represented by SEQ ID NO: 185, leucine at position 165 of the amino acid sequence represented by SEQ ID NO: 186, and leucine at position 164 of the amino acid sequence represented by SEQ ID NO: 187 are a common sequence between the polypeptide of the intracellular region of human 4-1BB and the polypeptide of the intracellular region of human CD3.

In the present specification, the “immunocompetent cell” means a cell responsible for immune functions in a living body. Examples of the immunocompetent cell can include: a lymphoid cell such as a T cell, a natural killer cell (NK cell), and a B cell; an antigen-presenting cell such as a monocyte, a macrophage, and a dendritic cell; and a granulocyte such as a neutrophil, an eosinophil, a basophil, and a mast cell. Specific examples thereof can preferably include a T cell derived from a mammal such as a human, a dog, a cat, a pig, or a mouse, preferably a human-derived T cell. The T cell can be obtained by isolation or purification from an immunocompetent cell infiltrating a body fluid such as blood or bone marrow fluid, a tissue of the spleen, the thymus, lymph node or the like, or a cancer tissue of primary tumor, metastatic tumor, cancerous ascites or the like. Alternatively, a T cell prepared from an ES cell or an iPS cell may be utilized. Examples of such a T cell can include an alpha-beta T cell, a gamma-delta T cell, a CD8⁺ T cell, a CD4⁺ T cell, a tumor-infiltrating T cell, a memory T cell, a naive T cell, and a NKT cell. The origin of the immunocompetent cell may be the same as or different from an administration subject. When the administration subject is a human, an autologous cell collected from a patient as the administration subject may be used as the immunocompetent cell, or any of other cells collected from a person other than the administration subject may be used as the immunocompetent cell. Specifically, the donor and the recipient may be the same or different and is preferably the same.

Examples of the administration subject can preferably include a mammal and a mammalian cell. Examples of the mammal can more preferably include a human, a mouse, a dog, a rat, a guinea pig, a rabbit, a bird, sheep, a pig, cattle, a horse, a cat, a monkey, and a chimpanzee, particularly preferably a human.

The present CAR is preferably used for ex vivo expression on the cell surface of the immunocompetent cell collected from a cancer patient in cancer treatment. In the case of using a T cell as the immunocompetent cell, examples of the peptide consisting of the transmembrane region and the immunocompetent cell activation signal transduction region fused with the C terminus of the transmembrane region in the present CAR can specifically include a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 185 to 187. Examples of the present CAR can specifically include CAR comprising single chain antibody selected from the group consisting of the single chain antibody described above in (1-3), the single chain antibody described above in (2-3), the single chain antibody described above in (1-3′-1), the single chain antibody described above in (1-3′-2), the single chain antibody described above in (1-3′-3), the single chain antibody described above in (2-3′-1), the single chain antibody described above in (2-3′-2), the single chain antibody described above in (2-3′-3), and the single chain antibody described above in (2-3′-4), and a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 185 to 187, fused with the C terminus of the single chain antibody.

Specifically, examples of the present CAR can include

CAR comprising the single chain antibody described above in (1-3), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 185, CAR comprising the single chain antibody described above in (1-3), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 186, CAR comprising the single chain antibody described above in (1-3), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 187, CAR comprising the single chain antibody described above in (1-3′-1), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 185, CAR comprising the single chain antibody described above in (1-3′-1), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 186, CAR comprising the single chain antibody described above in (1-3′-1), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 187, CAR comprising the single chain antibody described above in (1-3′-2), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 185, CAR comprising the single chain antibody described above in (1-3′-2), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 186, CAR comprising the single chain antibody described above in (1-3′-2), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 187, CAR comprising the single chain antibody described above in (1-3′-3), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 185, CAR comprising the single chain antibody described above in (1-3′-3), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 186, CAR comprising the single chain antibody described above in (1-3′-3), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 187, CAR comprising the single chain antibody described above in (2-3), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 185, CAR comprising the single chain antibody described above in (2-3), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 186, CAR comprising the single chain antibody described above in (2-3), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 187, CAR comprising the single chain antibody described above in (2-3′-1), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 185, CAR comprising the single chain antibody described above in (2-3′-1), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 186, CAR comprising the single chain antibody described above in (2-3′-1), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 187, CAR comprising the single chain antibody described above in (2-3′-2), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 185, CAR comprising the single chain antibody described above in (2-3′-2), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 186, CAR comprising the single chain antibody described above in (2-3′-2), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 187, CAR comprising the single chain antibody described above in (2-3′-3), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 185, CAR comprising the single chain antibody described above in (2-3′-3), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 186, CAR comprising the single chain antibody described above in (2-3′-3), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 187, CAR comprising the single chain antibody described above in (2-3′-4), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 185, CAR comprising the single chain antibody described above in (2-3′-4), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 186, and CAR comprising the single chain antibody described above in (2-3′-4), and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 187.

The present immunocompetent cell can be any immunocompetent cell expressing CAR. Since CAR usually does not occur naturally, the immunocompetent cell expresses foreign CAR, not endogenous CAR. The present immunocompetent cell preferably further expresses IL-7 and/or CCL19. When the immunocompetent cell is a cell found to not express IL-7 and/or CCL19, for example, a T cell, or when the immunocompetent cell is a cell, other than a T cell, low expressing IL-7 and/or CCL19, the present immunocompetent cell preferably expresses foreign IL-7 and/or CCL19.

The present immunocompetent cell can be prepared by introducing the present vector comprising the present CAR gene, and a vector comprising IL-7 and/or CCL19 gene to an immunocompetent cell. The introduction method can be any method for introducing DNA to mammalian cells. Examples thereof can include a method such as electroporation (Cytotechnology, 3, 133 (1990)), calcium phosphate method (Japanese unexamined Patent Application Publication No. 2-227075), lipofection (Proc. Natl. Acad. Sci. U.S.A., 84, 7413 (1987)), and viral infection method. Examples of such a viral infection method can include a method which involves transfecting a packaging cell such as a GP2-293 cell (manufactured by Takara Bio Inc.), a Plat-GP cell (manufactured by Cosmo Bio Co., Ltd.), a PG13 cell (ATCC CRL-10686), or a PA317 cell (ATCC CRL-9078) with a CAR expression vector (International Publication No. WO 2016/056228) and a packaging plasmid to prepare a recombinant virus, and infecting a T cell with the recombinant virus.

The present immunocompetent cell may be produced by incorporating a nucleotide encoding the present CAR and a nucleotide encoding IL-7 and/or CCL19 into the genome of a cell by use of a gene editing technique known in the art such that the nucleotides are expressible under the control of an appropriate promoter. Examples of the gene editing technique known in the art include a technique using endonuclease such as zinc finger nuclease, TALEN (transcription activator-like effector nuclease), or CRISPR (clustered regularly interspaced short palindromic repeat)-Cas system.

The present immunocompetent cell can be used in combination with an additional anticancer agent. Examples of the additional anticancer agent can include: an alkylating drug such as cyclophosphamide, bendamustine, ifosfamide, and dacarbazine; an antimetabolite such as pentostatin, fludarabine, cladribine, methotrexate, 5-fluorouracil, 6-mercaptopurine, and enocitabine; a molecular targeting drug such as rituximab, cetuximab, and trastuzumab; a kinase inhibitor such as imatinib, gefitinib, erlotinib, afatinib, dasatinib, sunitinib, and trametinib; a proteasome inhibitor such as bortezomib; a calcineurin inhibitory drug such as cyclosporin and tacrolimus; an anticancer antibiotic such as idarubicin and doxorubicin mitomycin C; a vegetable alkaloid such as irinotecan and etoposide; a platinum-containing drug such as cisplatin, oxaliplatin, and carboplatin; a hormone therapeutic such as tamoxifen and bicalutamide; and an immunosuppressive drug such as interferon, nivolumab, and pembrolizumab.

Examples of the method for “using the present immunocompetent cell in combination with the additional anticancer agent” can include a method using treatment with the additional anticancer agent followed by use of the present immunocompetent cell, a method using the present immunocompetent cell and the additional anticancer agent at the same time, and a method using treatment with the present immunocompetent cell followed by use of the additional anticancer agent. Use of the present immunocompetent cell in combination with the additional anticancer agent can further improve a therapeutic effect on a cancer and can also reduce their respective adverse reactions by decreasing their respective numbers of administration or doses.

The present antibody gene is not particularly limited as long as the antibody gene (nucleotide) encodes the present antibody. Examples thereof can include

(1-1D) an antibody gene comprising: a H chain CDR1 gene consisting of nucleotide residues 91 to 105 of a H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 111 (gene encoding the H chain CDR1 described above in (1-1)), or a degenerate codon engineered form of the H chain CDR1 gene; a H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 111 (gene encoding the H chain CDR2 described above in (1-1)), or a degenerate codon engineered form of the H chain CDR2 gene; and a H chain CDR3 gene consisting of nucleotide residues 295 to 324 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 111 (gene encoding the H chain CDR3 described above in (1-1)), or a degenerate codon engineered form of the H chain CDR3 gene; and a L chain CDR1 gene consisting of nucleotide residues 70 to 102 of a L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 112 (gene encoding the L chain CDR1 described above in (1-1)), or a degenerate codon engineered form of the L chain CDR1 gene; a L chain CDR2 gene consisting of nucleotide residues 148 to 168 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 112 (gene encoding the L chain CDR2 described above in (1-1)), or a degenerate codon engineered form of the L chain CDR2 gene; and a L chain CDR3 gene consisting of nucleotide residues 265 to 291 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 112 (gene encoding the L chain CDR3 described above in (1-1)), or a degenerate codon engineered form of the L chain CDR3 gene, (2-1D) an antibody gene comprising: a H chain CDR1 gene consisting of nucleotide residues 91 to 105 of a H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 115 (gene encoding the H chain CDR1 described above in (2-1)), or a degenerate codon engineered form of the H chain CDR1 gene; a H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 115 (gene encoding the H chain CDR2 described above in (2-1)), or a degenerate codon engineered form of the H chain CDR2 gene; and a H chain CDR3 gene consisting of nucleotide residues 295 to 321 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 115 (gene encoding the H chain CDR3 described above in (2-1)), or a degenerate codon engineered form of the H chain CDR3 gene; and a L chain CDR1 gene consisting of nucleotide residues 70 to 117 of a L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 116 (gene encoding the L chain CDR1 described above in (2-1)), or a degenerate codon engineered form of the L chain CDR1 gene; a L chain CDR2 gene consisting of nucleotide residues 163 to 183 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 116 (gene encoding the L chain CDR2 described above in (2-1)), or a degenerate codon engineered form of the L chain CDR2 gene; and a L chain CDR3 gene consisting of nucleotide residues 280 to 306 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 116 (gene encoding the L chain CDR3 described above in (2-1)), or a degenerate codon engineered form of the L chain CDR3 gene, (3-1D) an antibody gene comprising: a H chain CDR1 gene consisting of nucleotide residues 91 to 105 of a H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 119 (gene encoding the H chain CDR1 described above in (3-1)), or a degenerate codon engineered form of the H chain CDR1 gene; a H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 119 (gene encoding the H chain CDR2 described above in (3-1)), or a degenerate codon engineered form of the H chain CDR2 gene; and a H chain CDR3 gene consisting of nucleotide residues 295 to 315 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 119 (gene encoding the H chain CDR3 described above in (3-1)), or a degenerate codon engineered form of the H chain CDR3 gene; and a L chain CDR1 gene consisting of nucleotide residues 70 to 102 of a L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 120 (gene encoding the L chain CDR1 described above in (3-1)), or a degenerate codon engineered form of the L chain CDR1 gene; a L chain CDR2 gene consisting of nucleotide residues 148 to 168 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 120 (gene encoding the L chain CDR2 described above in (3-1)), or a degenerate codon engineered form of the L chain CDR2 gene; and a L chain CDR3 gene consisting of nucleotide residues 265 to 288 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 120 (gene encoding the L chain CDR3 described above in (3-1)), or a degenerate codon engineered form of the L chain CDR3 gene, (4-1D) an antibody gene comprising: a H chain CDR1 gene consisting of nucleotide residues 91 to 105 of a H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 123 (gene encoding the H chain CDR1 described above in (4-1)), or a degenerate codon engineered form of the H chain CDR1 gene; a H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 123 (gene encoding the H chain CDR2 described above in (4-1)), or a degenerate codon engineered form of the H chain CDR2 gene; and a H chain CDR3 gene consisting of nucleotide residues 298 to 330 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 123 (gene encoding the H chain CDR3 described above in (4-1)), or a degenerate codon engineered form of the H chain CDR3 gene; and a L chain CDR1 gene consisting of nucleotide residues 70 to 102 of a L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 124 (gene encoding the L chain CDR1 described above in (4-1)), or a degenerate codon engineered form of the L chain CDR1 gene; a L chain CDR2 gene consisting of nucleotide residues 148 to 168 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 124 (gene encoding the L chain CDR2 described above in (4-1)), or a degenerate codon engineered form of the L chain CDR2 gene; and a L chain CDR3 gene consisting of nucleotide residues 265 to 291 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 124 (gene encoding the L chain CDR3 described above in (4-1)), or a degenerate codon engineered form of the L chain CDR3 gene, (5-1D) an antibody gene comprising: a H chain CDR1 gene consisting of nucleotide residues 91 to 105 of a H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 127 (gene encoding the H chain CDR1 described above in (5-1)), or a degenerate codon engineered form of the H chain CDR1 gene; a H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 127 (gene encoding the H chain CDR2 described above in (5-1)), or a degenerate codon engineered form of the H chain CDR2 gene; and a H chain CDR3 gene consisting of nucleotide residues 298 to 321 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 127 (gene encoding the H chain CDR3 described above in (5-1)), or a degenerate codon engineered form of the H chain CDR3 gene; and a L chain CDR1 gene consisting of nucleotide residues 70 to 120 of a L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 128 (gene encoding the L chain CDR1 described above in (5-1)), or a degenerate codon engineered form of the L chain CDR1 gene; a L chain CDR2 gene consisting of nucleotide residues 166 to 186 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 128 (gene encoding the L chain CDR2 described above in (5-1)), or a degenerate codon engineered form of the L chain CDR2 gene; and a L chain CDR3 gene consisting of nucleotide residues 283 to 309 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 128 (gene encoding the L chain CDR3 described above in (5-1)), or a degenerate codon engineered form of the L chain CDR3 gene, (6-1D) an antibody gene comprising: a H chain CDR1 gene consisting of nucleotide residues 91 to 105 of a H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 131 (gene encoding the H chain CDR1 described above in (6-1)), or a degenerate codon engineered form of the H chain CDR1 gene; a H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 131 (gene encoding the H chain CDR2 described above in (6-1)), or a degenerate codon engineered form of the H chain CDR2 gene; and a H chain CDR3 gene consisting of nucleotide residues 295 to 318 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 131 (gene encoding the H chain CDR3 described above in (6-1)), or a degenerate codon engineered form of the H chain CDR3 gene; and a L chain CDR1 gene consisting of nucleotide residues 70 to 102 of a L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 132 (gene encoding the L chain CDR1 described above in (6-1)), or a degenerate codon engineered form of the L chain CDR1 gene; a L chain CDR2 gene consisting of nucleotide residues 148 to 168 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 132 (gene encoding the L chain CDR2 described above in (6-1)), or a degenerate codon engineered form of the L chain CDR2 gene; and a L chain CDR3 gene consisting of nucleotide residues 265 to 291 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 132 (gene encoding the L chain CDR3 described above in (6-1)), or a degenerate codon engineered form of the L chain CDR3 gene, (7-1D) an antibody gene comprising: a H chain CDR1 gene consisting of nucleotide residues 91 to 105 of a H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 135 (gene encoding the H chain CDR1 described above in (7-1)), or a degenerate codon engineered form of the H chain CDR1 gene; a H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 135 (gene encoding the H chain CDR2 described above in (7-1)), or a degenerate codon engineered form of the H chain CDR2 gene; and a H chain CDR3 gene consisting of nucleotide residues 295 to 315 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 135 (gene encoding the H chain CDR3 described above in (7-1)), or a degenerate codon engineered form of the H chain CDR3 gene; and a L chain CDR1 gene consisting of nucleotide residues 70 to 102 of a L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 136 (gene encoding the L chain CDR1 described above in (7-1)), or a degenerate codon engineered form of the L chain CDR1 gene; a L chain CDR2 gene consisting of nucleotide residues 148 to 168 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 136 (gene encoding the L chain CDR2 described above in (7-1)), or a degenerate codon engineered form of the L chain CDR2 gene; and a L chain CDR3 gene consisting of nucleotide residues 265 to 291 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 136 (gene encoding the L chain CDR3 described above in (7-1)), or a degenerate codon engineered form of the L chain CDR3 gene, (8-1D) an antibody gene comprising: a H chain CDR1 gene consisting of nucleotide residues 91 to 105 of a H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 139 (gene encoding the H chain CDR1 described above in (8-1)), or a degenerate codon engineered form of the H chain CDR1 gene; a H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 139 (gene encoding the H chain CDR2 described above in (8-1)), or a degenerate codon engineered form of the H chain CDR2 gene; and a H chain CDR3 gene consisting of nucleotide residues 295 to 315 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 139 (gene encoding the H chain CDR3 described above in (8-1)), or a degenerate codon engineered form of the H chain CDR3 gene; and a L chain CDR1 gene consisting of nucleotide residues 70 to 102 of a L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 140 (gene encoding the L chain CDR1 described above in (8-1)), or a degenerate codon engineered form of the L chain CDR1 gene; a L chain CDR2 gene consisting of nucleotide residues 148 to 168 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 140 (gene encoding the L chain CDR2 described above in (8-1)), or a degenerate codon engineered form of the L chain CDR2 gene; and a L chain CDR3 gene consisting of nucleotide residues 265 to 291 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 140 (gene encoding the L chain CDR3 described above in (8-1)), or a degenerate codon engineered form of the L chain CDR3 gene, (9-1D) an antibody gene comprising: a H chain CDR1 gene consisting of nucleotide residues 91 to 105 of a H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 143 (gene encoding the H chain CDR1 described above in (9-1)), or a degenerate codon engineered form of the H chain CDR1 gene; a H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 143 (gene encoding the H chain CDR2 described above in (9-1)), or a degenerate codon engineered form of the H chain CDR2 gene; and a H chain CDR3 gene consisting of nucleotide residues 298 to 330 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 143 (gene encoding the H chain CDR3 described above in (9-1)), or a degenerate codon engineered form of the H chain CDR3 gene; and a L chain CDR1 gene consisting of nucleotide residues 70 to 102 of a L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 144 (gene encoding the L chain CDR1 described above in (9-1)), or a degenerate codon engineered form of the L chain CDR1 gene; a L chain CDR2 gene consisting of nucleotide residues 148 to 168 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 144 (gene encoding the L chain CDR2 described above in (9-1)), or a degenerate codon engineered form of the L chain CDR2 gene; and a L chain CDR3 gene consisting of nucleotide residues 265 to 291 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 144 (gene encoding the L chain CDR3 described above in (9-1)), or a degenerate codon engineered form of the L chain CDR3 gene, (10-1D) an antibody gene comprising: a H chain CDR1 gene consisting of nucleotide residues 91 to 105 of a H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 147 (gene encoding the H chain CDR1 described above in (10-1)), or a degenerate codon engineered form of the H chain CDR1 gene; a H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 147 (gene encoding the H chain CDR2 described above in (10-1)), or a degenerate codon engineered form of the H chain CDR2 gene; and a H chain CDR3 gene consisting of nucleotide residues 295 to 327 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 147 (gene encoding the H chain CDR3 described above in (10-1)), or a degenerate codon engineered form of the H chain CDR3 gene; and a L chain CDR1 gene consisting of nucleotide residues 70 to 102 of a L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 148 (gene encoding the L chain CDR1 described above in (10-1)), or a degenerate codon engineered form of the L chain CDR1 gene; a L chain CDR2 gene consisting of nucleotide residues 148 to 168 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 148 (gene encoding the L chain CDR2 described above in (10-1)), or a degenerate codon engineered form of the L chain CDR2 gene; and a L chain CDR3 gene consisting of nucleotide residues 265 to 291 of the L chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 148 (gene encoding the L chain CDR3 described above in (10-1)), or a degenerate codon engineered form of the L chain CDR3 gene, and (11-1D) an antibody gene comprising: a H chain CDR1 gene consisting of nucleotide residues 91 to 105 of a H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 151 (gene encoding the H chain CDR1 described above in (11-1)), or a degenerate codon engineered form of the H chain CDR1 gene; a H chain CDR2 gene consisting of nucleotide residues 148 to 198 of the H chain V region gene consisting of the nucleotide sequence represented by SEQ ID NO: 151 (gene encoding the H chain CDR2 described above in (11-1)), or a degenerate codon engineered form of the H chain CDR2 gene; and a H chain CDR3 gene consisting of nucleotide residues 295 to 324 of the H chain V region consisting of the nucleotide sequence represented by SEQ ID NO: 151 (gene encoding the H chain CDR3 described above in (11-1)), or a degenerate codon engineered form of the H chain CDR3 gene.

Further examples of the present antibody gene can include

(1-2D) an antibody gene comprising a H chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 111 (gene encoding a H chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 7), and a L chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 112 (gene encoding a L chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 8), (2-2D) an antibody gene comprising a H chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 115 (gene encoding a H chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 17), and a L chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 116 (gene encoding a L chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 18), (3-2D) an antibody gene comprising a H chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 119 (gene encoding a H chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 27), and a L chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 120 (gene encoding a L chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 28), (4-2D) an antibody gene comprising a H chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 123 (gene encoding a H chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 37), and a L chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 124 (gene encoding a L chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 38), (5-2D) an antibody gene comprising a H chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 127 (gene encoding a H chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 47), and a L chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 128 (gene encoding a L chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 48), (6-2D) an antibody gene comprising a H chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 131 (gene encoding a H chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 57), and a L chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 132 (gene encoding a L chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 58), (7-2D) an antibody gene comprising a H chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 135 (gene encoding a H chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 67), and a L chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 136 (gene encoding a L chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 68), (8-2D) an antibody gene comprising a H chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 139 (gene encoding a H chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 77), and a L chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 140 (gene encoding a L chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 78), (9-2D) an antibody gene comprising a H chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 143 (gene encoding a H chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 87), and a L chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 144 (gene encoding a L chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 88), (10-2D) an antibody gene comprising a H chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 147 (gene encoding a H chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 97), and a L chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 148 (gene encoding a L chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 98), and (11-2D) an antibody gene comprising a H chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 151 (gene encoding a H chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 107), and a L chain variable region gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 152 (gene encoding a L chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 108).

Particularly, examples of the present antibody gene can specifically include

(1-4D) an antibody gene comprising a H chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 113 (gene encoding a H chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 9), and a L chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 114 (gene encoding a L chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 10), (2-4D) an antibody gene comprising a H chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 117 (gene encoding a H chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 19), and a L chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 118 (gene encoding a L chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 20), (3-4D) an antibody gene comprising a H chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 121 (gene encoding a H chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 29), and a L chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 122 (gene encoding a L chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 30), (4-4D) an antibody gene comprising a H chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 125 (gene encoding a H chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 39), and a L chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 126 (gene encoding a L chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 40), (5-4D) an antibody gene comprising a H chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 129 (gene encoding a H chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 49), and a L chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 130 (gene encoding a L chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 50), (6-4D) an antibody gene comprising a H chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 133 (gene encoding a H chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 59), and a L chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 134 (gene encoding a L chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 60), (7-4D) an antibody gene comprising a H chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 137 (gene encoding a H chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 69), and a L chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 138 (gene encoding a L chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 70), (8-4D) an antibody gene comprising a H chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 141 (gene encoding a H chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 79), and a L chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 142 (gene encoding a L chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 80), (9-4D) an antibody gene comprising a H chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 145 (gene encoding a H chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 89), and a L chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 146 (gene encoding a L chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 90), (10-4D) an antibody gene comprising a H chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 149 (gene encoding a H chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 99), and a L chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 150 (gene encoding a L chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 100), and (11-4D) an antibody gene comprising a H chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 153 (gene encoding a H chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 109), and a L chain gene consisting of a nucleotide sequence having at least 80% or higher sequence identity to the nucleotide sequence represented by SEQ ID NO: 154 (gene encoding a L chain consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 110).

The present CAR gene is not particularly limited as long as the gene (nucleotide) encodes the present CAR. Examples thereof can specifically include

(1-3D) a CAR gene comprising a gene encoding the single chain antibody described above in (1-3), or a degenerate codon engineered form of the gene, (2-3D) a CAR gene comprising a gene encoding the single chain antibody described above in (2-3), or a degenerate codon engineered form of the gene, (3-3D) a CAR gene comprising a gene encoding the single chain antibody described above in (3-3), or a degenerate codon engineered form of the gene, (4-3D) a CAR gene comprising a gene encoding the single chain antibody described above in (4-3), or a degenerate codon engineered form of the gene, (5-3D) a CAR gene comprising a gene encoding the single chain antibody described above in (5-3), or a degenerate codon engineered form of the gene, (6-3D) a CAR gene comprising a gene encoding the single chain antibody described above in (6-3), or a degenerate codon engineered form of the gene, (7-3D) a CAR gene comprising a gene encoding the single chain antibody described above in (7-3), or a degenerate codon engineered form of the gene, (8-3D) a CAR gene comprising a gene encoding the single chain antibody described above in (8-3), or a degenerate codon engineered form of the gene, (9-3D) a CAR gene comprising a gene encoding the single chain antibody described above in (9-3), or a degenerate codon engineered form of the gene, (10-3D) a CAR gene comprising a gene encoding the single chain antibody described above in (10-3), or a degenerate codon engineered form of the gene, (11-3D) a CAR gene comprising a gene encoding the single chain antibody described above in (11-3), or a degenerate codon engineered form of the gene, (1-3′-1D) a CAR gene comprising a gene encoding the single chain antibody described above in (1-3′-1), or a degenerate codon engineered form of the gene, (1-3′-2D) a CAR gene comprising a gene encoding the single chain antibody described above in (1-3′-2), or a degenerate codon engineered form of the gene, (1-3′-3D) a CAR gene comprising a gene encoding the single chain antibody described above in (1-3′-3), or a degenerate codon engineered form of the gene, (2-3′-1D) a CAR gene comprising a gene encoding the single chain antibody described above in (2-3′-1), or a degenerate codon engineered form of the gene, (2-3′-2D) a CAR gene comprising a gene encoding the single chain antibody described above in (2-3′-2), or a degenerate codon engineered form of the gene, (2-3′-3D) a CAR gene comprising a gene encoding the single chain antibody described above in (2-3′-3), or a degenerate codon engineered form of the gene, and (2-3′-4D) a CAR gene comprising a gene encoding the single chain antibody described above in (2-3′-4), or a degenerate codon engineered form of the gene.

In the present specification, the phrase “at least 80% or higher identity” means that the identity is 80% or higher, preferably 85% or higher, more preferably 88% or higher, further preferably 90% or higher, still further preferably 93% or higher, particularly preferably 95% or higher, particularly more preferably 98% or higher, most preferably 100%.

In the present specification, the term “identity” means the degree of similarity between polypeptide or polynucleotide sequences (this degree is determined by matching a query sequence to another sequence, preferably of the same type (nucleic acid or protein sequence)). Examples of a preferred computer program method for calculating and determining the “identity” include, but are not limited to, GCG BLAST (Basic Local Alignment Search Tool) (Altschul et al., J. Mol. Biol. 1990, 215: 403-410; Altschul et al., Nucleic Acids Res. 1997, 25: 3389-3402; and Devereux et al., Nucleic Acid Res. 1984, 12: 387), BLASTN 2.0 (Gish W., http://blast.wustl.edu,1996-2002), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 1988, 85: 2444-2448), and GCG GelMerge which determines and aligns a pair of the longest overlapping contigs (Wibur and Lipman, SIAM J. Appl. Math. 1984, 44: 557-567; and Needleman and Wunsch, J. Mol. Biol. 1970, 48: 443-453).

In the present specification, the “amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: X” is, in other words, an “amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: X by the deletion, substitution, insertion, and/or addition of 0, 1 or several amino acid residues” and has functions equivalent to those of the amino acid sequence represented by SEQ ID NO: X. In this context, the “amino acid sequence derived by the deletion, substitution, insertion, and/or addition of 1 or several amino acid residues” means an amino acid sequence in which amino acid residues have been deleted, substituted, inserted, and/or added, for example, within the range of 1 to 30 residues, preferably within the range of 1 to 20 residues, more preferably within the range of 1 to 15 residues, further preferably within the range of 1 to 10 residues, further preferably within the range of 1 to 5 residues, further preferably within the range of 1 to 3 residues, further preferably within the range of 1 or 2 residues. The mutation treatment of these amino acid residues can be performed by an arbitrary method known to those skilled in the art such as chemical synthesis, a gene engineering approach, or mutagenesis.

The promoter in the present vector can be any region that starts the transcription of mRNA encoded by the present antibody gene located downstream of the promoter. The promoter usually comprises a transcription start site (TSS).

The type of the promoter or the vector in the present vector can be appropriately selected according to the type of a host cell (or a host organism) to which the present vector is introduced.

The host cell can express the present antibody by the transcription of the present antibody gene, or can express the present CAR by the transcription of mRNA of the present CAR gene. In the case of introducing a “vector comprising the present antibody gene” as the present vector, a yeast, a mammalian cell, an insect cell, or a plant cell given below can be used as the host cell. In the case of introducing a “vector comprising the present CAR gene” as the present vector, the immunocompetent cell described above can be used as the host cell.

In the case of using a yeast (e.g., Saccharomyces cerevisiae and Schizosaccharomyces pombe) as the host cell, examples of the present vector can include a vector such as YEP13 (ATCC37115), YEp24 (ATCC37051), and YCp50 (ATCC37419), and a vector derived from the vector. Examples of the promoter can include glycolysis gene (e.g., hexose kinase gene) promoter, PHOS promoter, PGK promoter, GAP promoter, ADH promoter, gall promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, and CUP1 promoter.

In the case of using a mammalian cell (e.g., a human-derived Namalwa cell, a monkey-derived COS cell, a Chinese hamster ovary-derived CHO cell, and a human- or mouse-derived T cell) as the host cell and using a vector comprising the antibody gene as the present vector, examples of the present vector can include a vector such as pcDNAI, pcDM8 (manufactured by Funakoshi Co., Ltd.), pAGE107 (Japanese unexamined Patent Application Publication No. 3-22979; and Cytotechnology, 3, 133, (1990)), pAS3-3 (Japanese unexamined Patent Application Publication No. 2-227075), pCDM8 (Nature, 329, 840, (1987)), pcDNAI/Amp (manufactured by Invitrogen Corp.), pREP4 (manufactured by Invitrogen Corp.), pAGE103 (J. Biochemistry, 101, 1307 (1987)), and pAGE210, and a vector derived from the vector. On the other hand, in the case of using a mammalian cell (e.g., the human-derived immunocompetent cell described above) as the host cell and using a vector comprising the CAR gene as the present vector, examples of the present vector can include a retrovirus vector such as a pMSGV vector (Tamada k et al., Clin Cancer Res 18: 6436-6445 (2002)) and a pMSCV vector (manufactured by Takara Bio Inc.), and a vector derived from the vector.

Examples of the promoter in the present vector can include cytomegalovirus (CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus promoter, metallothionein promoter, heat shock promoter, SRα promoter, NFAT promoter, and HIF promoter.

In the case of using an insect cell (e.g., a Sf9 cell and a Sf21 cell which are Spodoptera frugiperda ovarian cells, and a High5 cell which is a Trichoplusia ni ovarian cell) as the host cell, examples of the present vector can include a transfer vector for use in recombinant baculovirus preparation methods, specifically, a vector such as pVL1392, pVL1393, and pBlueBacIII (all manufactured by Invitrogen Corp.), and a vector derived from the vector. Examples of the promoter can include polyhedrin promoter and p10 promoter.

In the case of using a plant cell (e.g., tobacco, potato, tomato, carrot, soybean, rapeseed, alfalfa, rice, wheat, and barley cells) as the host cell, examples of the expression vector can include a vector such as Ti plasmid and tobacco mosaic virus vector, and a vector derived from the vector. Examples of the promoter can include cauliflower mosaic virus (CaMV) 35S promoter and rice actin 1 promoter.

The present vector preferably further comprises the nucleotide sequences of an enhancer region and a ribosome binding site (RBS) for further enhancing gene expression efficiency, and further comprises a drug resistance gene (e.g., spectinomycin resistance gene, chloramphenicol resistance gene, tetracycline resistance gene, kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, hygromycin resistance gene, blasticidin resistance gene, and geneticin resistance gene) appropriate for the type of the host cell for screening for the present host cell. The enhancer region is usually arranged upstream of the promoter, and RBS is usually arranged between the promoter and the present gene. The nucleotide sequence of the present antibody gene to be incorporated into the present vector may be subjected to the optimization of a codon sequence according to the host cell for expression. The present vector can be prepared by a method known in the art using a gene recombination technique.

The present host cell can be obtained by introducing the present vector to the host cell (transfecting the host cell therewith) by a method appropriate for the type of the host cell.

In the case of using the yeast described above as the host cell, the method for introducing the present vector to the yeast can be any method for introducing DNA to the yeast. Examples thereof can include a method such as electroporation (Methods Enzymol., 194, 182 (1990)), spheroplast method (Proc. Natl. Acad. Sci. U.S.A, 84, 1929 (1978)), and lithium acetate method (J. Bacteriology, 153, 163 (1983)).

In the case of using the mammalian cell described above as the host cell, the method for introducing the present vector to the mammalian cell can be any method for introducing DNA to the mammalian cell. Examples thereof can include a method such as electroporation (Cytotechnology, 3, 133 (1990)), calcium phosphate method (Japanese unexamined Patent Application Publication No. 2-227075), lipofection (Proc. Natl. Acad. Sci. U.S.A., 84, 7413 (1987)), and viral infection method, as mentioned above. Examples of such a viral infection method can include a method which involves transfecting a packaging cell such as a GP2-293 cell (manufactured by Takara Bio Inc.), a Plat-GP cell (manufactured by Cosmo Bio Co., Ltd.), a PG13 cell (ATCC CRL-10686), or a PA317 cell (ATCC CRL-9078) with a CAR expression vector (International Publication No. WO 2016/056228) and a packaging plasmid to prepare a recombinant virus, and infecting a T cell with the recombinant virus, as mentioned above.

In the case of using the insect cell described above as the host cell, examples of the method for introducing the present vector to the insect cell can include a method which involves cotransfecting the insect cell with the present vector (transfer vector) and baculovirus-derived genomic DNA to prepare a recombinant baculovirus, according to a method described in “Current Protocols in Molecular Biology”, “Baculovirus Expression Vectors, A Laboratory Manual, W.H. Freeman and Company, New York (1992)”, “Bio/Technology, 6, 47 (1988)”, etc. Examples of such a cotransfection method can include a method such as calcium phosphate method (Japanese unexamined Patent Application Publication No. 2-227075) and lipofection (Proc. Natl. Acad. Sci. U.S.A., 84, 7413 (1987).

In the case of using the plant cell described above as the host cell, examples of the method for introducing the present vector to the plant cell can include a method such as a method using Agrobacterium (Japanese unexamined Patent Application Publication Nos. 59-140885 and 60-70080), electroporation (Japanese unexamined Patent Application Publication No. 60-251887), and a method using a particle gun (gene gun) (Japanese Patent Nos. 2606856 and 2517813).

The present antibody can be obtained by culturing the present host cell obtained by the method mentioned above in a culture solution appropriate for the host cell.

A transgenic animal, such as a mouse, cattle, a goat, sheep, a chicken, or a pig, in which the present antibody gene (the present vector) has been incorporated is prepared by use of a transgenic animal preparation technique, and an antibody derived from the present antibody gene can also be produced in a large amount from the blood, milk, or the like of the transgenic animal.

Nonhuman animals (e.g., mice and rats) are immunized with a substance comprising a human-derived GPC3 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 155 (GPC3 polypeptide antigen). A phage library of scFv genes is prepared by a phage display method. The present scFv can be obtained by a biopanning method using the GPC3 polypeptide antigen and/or a cell line expressing the GPC3 polypeptide antigen (preferably a cell line expressing no endogenous GPC3), and further, preferably, a competitor C-terminal polypeptide of GPC3 consisting of the amino acid sequence represented by SEQ ID NO: 159. From the nonhuman animals thus immunized with the antigen, antibody-producing hybridomas are prepared by use of a cell fusion technique. A culture supernatant containing the present antibody can also be obtained through screening by ELISA using a plate in which the antigen has been immobilized on a solid phase. The present antibody can be separated and purified from the culture supernatant by use of an antibody purification technique known in the art.

The present detection method can be any method comprising the step of detecting GPC3 localized on a cell membrane (anchored on a cell membrane) in a sample (e.g., blood, a tissue, and urine) using the present antibody. Specific examples of the detection method can include immunofluorescent staining, Western blotting, and ELISA using the present antibody.

The present kit for detection is a kit comprising the present antibody or a labeled form thereof and is limited by the purpose of “detecting GPC3”. The kit usually comprises components generally used in this kind of kit, for example, a carrier, a pH buffering agent, and a stabilizer as well as an attached document such as a manual and an instruction for detecting GPC3.

The organism species of GPC3 to be detected in the present detection method or the present kit for detection may be a nonhuman animal such as a mouse or a rat and is usually a human.

Examples of the labeling material for the labeled form of the present antibody can include: an enzyme such as peroxidase (e.g., horseradish peroxidase [HRP]), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apo-glucose oxidase, urease, luciferase and acetylcholinesterase; a fluorescent material such as fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl chloride and tetramethylrhodamine isothiocyanate; a fluorescence protein such as green fluorescence protein (GFP), cyan fluorescence protein (CFP), blue fluorescence protein (BFP), yellow fluorescence protein (YFP), red fluorescence protein (RFP) and luciferase; a radioisotope such as 3H, ¹⁴C, ¹²⁵I and ¹³¹I; biotin; avidin; and a chemiluminescence material.

References, such as scientific literatures, patents, and patent applications, cited herein are incorporated herein by reference in their entirety to the same extent as if each individual reference was specifically described. The present application claims the priority based on Japanese Patent Application No. 2017-001732 (filed on Jan. 10, 2017), the contents of which are incorporated herein by reference in their entirety.

Hereinafter, the present invention will be described more specifically with reference to Examples. However, the technical scope of the present invention is not limited by these examples.

Example 1 1. Preparation of Novel Anti-GPC3 Antibody Recognizing N-Terminal Polypeptide of Human GPC3 SUMMARY

SKG/Jcl mice were used as animals to be immunized for preparing an anti-human GPC3 antibody, and full-length human GPC3 protein was used as an immunizing antigen. The SKG/Jcl mice were autoimmune disease model mice that spontaneously develop rheumatoid arthritis and are known to produce antibodies in response even to self-components depending on aging or a rearing environment. Meanwhile, GPC3 is highly homologous between humans and mice and is usually less likely to cause antibody production even by the immunization of normal mice. Therefore, the SKG/Jcl mice were used as animals to be immunized. A scFv phage library was prepared from cDNA derived from B cells of the mice immunized with GPC3, and an anti-human GPC3 antibody was isolated by the application of the phage display method.

Although the antiserum of immunized mice contains many types of antibodies, it is necessary to select mice producing antibodies having specificity for the N-terminal polypeptide of GPC3 by excluding mice producing antibodies low specific for GPC3 or antibodies recognizing the C-terminal polypeptide of GPC3. Accordingly, mouse individuals that exhibited the production of an antibody specifically binding to the N-terminal polypeptide of GPC3 were selected by use of ELISA and FCM. Specifically, cDNA was synthesized by reverse transcription reaction from total RNA of the B cells derived from the immunized mice, and antibody genes were amplified to prepare an antibody gene library. A scFv phage library was constructed from the antibody gene library, and E. coli was infected with the library so that E. coli expressed scFv, followed by biopanning using recombinant GPC3, the GPC3-expressing cell line, and the C-terminal polypeptide of GPC3 to enrich phages expressing the target scFv, i.e., an antibody against the N-terminal polypeptide of GPC3. In order to further analyze the obtained scFv for binding specificity for GPC3 in cells, i.e., GPC3 localized on (bound to) a cell membrane (membrane-bound GPC3) via a GPI (glycosylphosphatidylinositol) anchor, verification was made by use of cell based-ELISA and FCM. Furthermore, the nucleotide sequences of H chain and L chain variable regions of clones having binding specificity were sequenced, and the nucleotide sequences of the anti-GPC3 antibodies produced by the B cells derived from the immunized mice were determined on the basis of these sequences. Finally, the mammalian display method which involved expressing the N-terminal polypeptide fragment and the C-terminal polypeptide fragment of GPC3 on cell surface was used to confirm that the epitope for the scFv was the N-terminal polypeptide fragment of GPC3. Hereinafter, detailed methods and results will be shown.

1-1 Material and Method [Cell Culture]

A JHH7 cell line, a HepG2 cell line, and a SK-Hep-1 cell line forced to express full-length human GPC3 (hereinafter, also referred to as a “GPC3-expressing cell line”) were used as human GPC3-expressing cells to perform the biopanning and screening of an anti-GPC3 antibody. The JHH7 cell line is a GPC3-expressing cell line derived from hepatocellular carcinoma, and the cells constitutively express GPC3 bound to a cell membrane (membrane-bound GPC3) via a GPI (glycosylphosphatidylinositol) anchor. On the other hand, the HepG2 cell line is a GPC3-expressing cell line derived from hepatocellular carcinoma, as in the JHH7 cell line, but is a cell line in which the expression of secretory GPC3 that is not bound to a cell membrane is dominant over membrane-bound GPC3. The Sk-Hep-1 cell line is a hepatocellular carcinoma-derived cell line expressing no GPC3. Hence, a cell line expressing only membrane-bound full-length GPC3 or membrane-bound GPC3 having a partial length deficient in a portion of exons can be prepared by forced expression.

The culture of 4 types of cell lines (JHH7 cell line, HepG2 cell line, GPC3-expressing cell line, and human embryonic kidney epithelium-derived 293T cell line) was performed under conditions of 37° C. and 5% CO₂ in a DMEM culture solution (manufactured by Sigma-Aldrich Co. LLC) containing 10% FBS (manufactured by Gibco/Thermo Fisher Scientific Inc.) and 1% penicillin-streptomycin (manufactured by Gibco/Thermo Fisher Scientific Inc.) (hereinafter, simply referred to as a “DMEM culture solution”). The culture of a CHO-K1 cell line was performed under conditions of 37° C. and 5% CO₂ in a Ham's F12 culture solution (manufactured by Sigma-Aldrich Co. LLC) containing 10% FBS (manufactured by Gibco/Thermo Fisher Scientific Inc.).

[Immunizing Antigen]

C-terminally 6×His-tagged recombinant GPC3 (manufactured by R & D Systems Inc.) was adjusted to 0.1 mg/mL with PBS and mixed with an artificial adjuvant TiterMax Gold (manufactured by TiterMax USA, Inc.) or CFA (Freund's Adjuvant Complete) (F5881, manufactured by Sigma-Aldrich Co. LLC) in equal amounts to prepare an emulsion, which was then used as an initial immunizing antigen. Recombinant GPC3 was adjusted to a concentration from 10 to 100 μg/mL with PBS and used as the second or later immunizing antigens.

[Preparation of GPC3-Expressing Cell Line]

A gene encoding full-length human GPC3 consisting of the amino acid sequence represented by SEQ ID NO: 157 (full-length human GPC3 gene consisting of the nucleotide sequence represented by SEQ ID NO: 160) was inserted to a pcDNA3.1 vector (manufactured by Thermo Fisher Scientific Inc.) to prepare a GPC3 expression vector. A SK-Hep-1 cell line was transfected with the GPC3 expression vector according to a standard method and then cultured in a DMEM culture solution containing G418 (manufactured by Roche Diagnostics K.K.) to establish a SK-Hep-1 cell line stably expressing full-length GPC3 (GPC3-expressing cell line).

[Immunization of Mouse]

SKG/Jcl mice (CLEA Japan, Inc., 8-week-old female, SPF) were used as animals to be immunized, and immunized through footpads with recombinant GPC3 a total of 4 times on 1-week intervals. On 5 weeks from the start of immunization, blood was collected, and serum was prepared according to a standard method and used as a specimen for the confirmation of an antibody titer.

[Serum Antibody Titer of Antiserum Using ELISA]

In order to confirm the response of the immunized mice to produce an anti-GPC3 antibody, a serum antibody titer was measured by use of antigen-immobilized ELISA. 0.5 or 2 μg/mL recombinant GPC3 was added at 50 μL/well to a 96-well microplate (manufactured by Nalge Nunc International), and the plate was incubated at room temperature for 1 hour or at 4° C. for 12 hours. Then, 2% Block ACE (manufactured by DS Pharma Biomedical Co., Ltd.) was added at 200 μL/well to perform blocking treatment. The serum derived from the GPC3-immunized mice was serially diluted from 100-fold to 16500-fold with 0.1% Block ACE/PBS solution. Each diluted serum sample was added at 50 μL/well, and the plate was incubated at room temperature for 2 hours to perform antigen-antibody reaction treatment. After washing of the wells with a Tween 20-containing PBS (PBST) solution, goat anti-mouse IgG (manufactured by Jackson ImmunoResearch Laboratories Inc.) conjugated with 2 μg/mL peroxidase was added thereto, and the plate was incubated at room temperature for 2 hours to perform secondary antibody reaction treatment. After washing of the well five times with a PBST solution, moisture was removed, and a TMB substrate (manufactured by Thermo Fisher Scientific Inc.) was then added at 50 μL/well to perform color reaction. 15 minutes later, the color reaction was terminated by the addition of 0.18 M sulfuric acid at 50 μL/well, followed by the measurement of absorbance at 450 nm and 540 nm using a plate reader (manufactured by Bio-Rad Laboratories, Inc.). Quantification was performed using a corrected value obtained by subtracting the measurement value of 540 nm from the measurement value of 450 nm.

[Specificity of Antibody in Antiserum Using FCM]

In order to further confirm the specific binding activity of the antiserum against membrane-bound GPC3 as to the immunized mice, the mouse serum diluted 100-fold and 5×10³ cells of the GPC3-expressing cell line were mixed and incubated for 30 minutes on ice. A FACS buffer (1% BSA/PBS solution) was added thereto, and the mixture was centrifuged to remove a supernatant. Then, 100 μL of 1 μg/mL goat anti-mouse IgG (H+L) Alexa Fluor 488 (manufactured by Thermo Fisher Scientific Inc.) was added as a secondary antibody, and the mixture was incubated for 30 minutes on ice to perform secondary antibody reaction treatment. The detection of Alexa Fluor 488 and the measurement of a fluorescence level were performed using a flow cytometer (FACSCanto) (manufactured by BD Biosciences).

[Preparation of scFv Phage Library]

B cells-derived total RNA was extracted according to a standard method as to the mice shown to produce an antibody binding to membrane-bound GPC3 by the method described above in the section [Flow cytometer]. RT-PCR with the total RNA as a template was performed according to a standard method to prepare cDNA. Antibody H chain and L chain variable region genes were amplified by PCR. A nucleotide sequence encoding a fusion protein of scFv having the H chain and L chain variable regions linked via a flexible linker, and coat protein g3p (cp3) of fibrous bacteriophage M13 was inserted to the multicloning site of a pTZ19R phagemid vector to prepare a scFv expression vector. The scFv library size was calculated from the transformation efficiency of an E. coli DH12S strain (manufactured by Invitrogen Corp.). The transformed DH12S strain was infected with a helper phage M13K07 (manufactured by Invitrogen Corp.) to prepare a phage library expressing scFv.

[Biopanning and Cloning of Phage scFv]

The biopanning of phage scFv using a combination of recombinant GPC3 immobilized on Dynabeads His-Tag Isolation & Pulldown magnetic beads (manufactured by VERITAS Corp.) via 6×His tag, and the GPC3-expressing cell line as a bait was performed according to the method described in a document such as “J Mol Biol. 1991 Dec. 5; 222 (3): 581-97”, “J Med Virol. 2007 June; 79 (6): 852-62”, “Proc Natl Acad Sci USA. 2008 May 20; 105 (20): 7287-92”, or “JOURNAL OF VIROLOGY, April 2004, p. 3325-3332 Vol. 78, No. 7”. In each round (step) of biopanning consisting of types of series (A to E series) (see FIG. 1), an aliquot of polyclonal phage antibodies was sampled. In order to confirm the binding specificity of scFv, antigen-immobilized ELISA was performed according to the method described above in the section [Serum antibody titer of antiserum using ELISA] (method using the culture supernatant of E. coli containing a phage instead of the serum), while cell-based ELISA was performed according to the method described below in the section [Screening of scFv by cell-based ELISA]. Each step of this biopanning was devised so as not to select a scFv phage binding to the same portion as the C-terminal epitope of GPC3 recognized by existing antibodies, by binding in advance the existing anti-GPC3 antibodies GC33 (manufactured by Chugai Pharmaceutical Co., Ltd.) and GC199 (manufactured by Chugai Pharmaceutical Co., Ltd.) to the bait. Specifically, this competition method enables selective panning of a novel antibody recognizing a GPC3 epitope different from that for the existing anti-GPC3 antibodies. E. coli DH12S was transformed with the phages enriched by biopanning and inoculated to an LB agarose agar medium to separate single colonies. The E. coli was further cultured in a small-scale LB liquid medium, followed by the extraction and purification of plasmids. The purified plasmids were subjected to DNA sequencing to determine the nucleotide sequences of scFv H chain and L chain variable regions.

[Screening of scFv by FCM]

100 μL of the culture supernatant in which scFv phages were secreted was added to a GPC3-expressing cell line (5×10⁵ cells per sample) and mixed therewith, and the mixture was then incubated for 30 minutes on ice. A FACS buffer (1% BSA/PBS solution) was added thereto, and the mixture was centrifuged and washed. Then, 1 μg/mL anti-mouse antibody-Alexa 488 (manufactured by Thermo Fisher Scientific Inc.) was added thereto as a secondary antibody, and the mixture was incubated for 30 minutes on ice. Then, the fluorescent staining of the cells was measured using a flow cytometer (FACSCanto, manufactured by BD Biosciences).

[Screening of scFv by Cell-Based ELISA]

After removal of a DMEM culture solution from a 96-well microplate in which 2×10³ GPC3-expressing cells were attached per well, 2% BSA-PBS solution was added for the purpose of preventing the nonspecific binding of scFv to the cells or the plate, and the plate was incubated for minutes on ice. Then, 100 μL of the culture supernatant of E. coli in which scFv phages were secreted was added to each well, and the plate was incubated for 45 minutes on ice. Then, 5 μg/mL rabbit anti-cp3 antibody (manufactured by Medical & Biological Laboratories Co., Ltd.) against cp3 fused on the C-terminal side of scFv was added at 100 μL per well, and the plate was further incubated for 45 minutes on ice. A HRP-labeled anti-rabbit IgG antibody (manufactured by Medical & Biological Laboratories Co., Ltd.) diluted 5000-fold was added at 100 μL per well as a tertiary antibody for anti-cp3 antibody detection, and the plate was incubated for 45 minutes on ice. Then, o-phenylenediamine (OPD) and hydrogen peroxide were added as substrates of HRP for color development. Quantification was performed using a numeric value obtained by subtracting absorbance at 620 nm as a background from absorbance at 492 nm. When cell-based ELISA was carried out using an antibody already converted to an IgG type antibody, not scFv, a HRP-labeled anti-mouse IgG antibody (manufactured by Medical & Biological Laboratories Co., Ltd.) diluted 2000-fold was used as a secondary antibody for the detection of the IgG type antibody instead of the anti-cp3 antibody and the HRP-labeled anti-rabbit IgG antibody among the conditions described above.

[Determination of Variable Region Gene Sequences of scFv]

The variable region gene sequences of phage scFv binding to membrane-bound GPC3 were decoded in a sequencer (CEQ2000XL, manufactured by Beckman Coulter, Inc.) using a T7 primer (primer consisting of the nucleotide sequence represented by SEQ ID NO: 176), which is a universal primer, and a cp3R primer (primer consisting of the nucleotide sequence represented by SEQ ID NO: 177) as a forward primer for H chain V region (V_(H)) decoding and a reverse primer for L chain V region (V_(L)) decoding, respectively.

[Preparation of Cell Line for Use in Antibody Epitope Mapping]

In order to identify an epitope for the cloned scFv, the mammalian display method was applied. A gene consisting of human GPC3 exons 1 to 7 and encoding a GPC3 N-terminal fragment (polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 155), and a gene consisting of human GPC3 exons 8 and 9 and encoding a GPC3 C-terminal fragment (polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 156) were amplified by PCR and each inserted to the multicloning site (MSC) of a pDisplay expression vector (manufactured by Thermo Fisher Scientific Inc.). The pDisplay expression vector is an expression vector capable of fusing a transmembrane domain of platelet-derived growth factor receptor (PDGFR) to the C terminus of the target protein and displaying the fusion product on the cell surface of arbitrary mammalian cells. Also, the pDisplay expression vector is constituted so as to add a HA tag to the N terminus of the target protein and to add a myc tag to the C terminus of the PDGFR. The pDisplay expression vector for expressing the GPC3 N-terminal fragment or the GPC3 C-terminal fragment was gene-transferred to a SK-Hep-cell line or a 293T cell line, and a cell line expressing the GPC3 N-terminal fragment or the GPC3 C-terminal fragment on the cell surface (GPC3 N-terminal fragment-expressing cell line and GPC3 C-terminal fragment-expressing cell line) was isolated and used in the epitope mapping of scFv.

[Antibody Epitope Mapping by FCM]

The GPC3 N-terminal fragment-expressing cell line, the GPC3 C-terminal fragment-expressing cell line, and the GPC3-expressing cell line (5×10³ cells each per sample) were each mixed with 100 μL of the culture supernatant in which scFv phages were secreted, and the mixture was incubated for 30 minutes on ice. A FACS buffer (1% BSA/PBS solution) was added thereto, and the mixture was centrifuged and washed. Then, 1 μg/mL anti-mouse antibody-Alexa 488 (manufactured by Thermo Fisher Scientific Inc.) was added thereto as a secondary antibody, and the mixture was incubated for 30 minutes on ice. Then, the fluorescent staining of the cells was measured using a flow cytometer (FACSCanto, manufactured by BD Biosciences).

[Construction of Recombinant IgG Expression Vector]

In order to convert scFv to IgG, an expression vector of Mammalian PowerExpress system (manufactured by Toyobo Co., Ltd.) was used. A nucleotide sequence encoding a fusion protein of the H chain variable region of scFv and a mouse IgG2a H chain-derived constant region was inserted to MSC of a pEH1.1 vector (pEH1.1-H). Also, a nucleotide sequence encoding a fusion protein of the L chain variable region of scFv and a mouse IgG2a L chain-derived constant region was inserted to MSC of a pELX2.2 vector (pEH2.2-L). Then, a polynucleotide fragment from EF1α promoter to the L chain gene was excised from pEH2.2-L with restriction enzymes (BglII and SalI) and ligated with pEH1.1-H treated with restriction enzymes (BglII and SalI) to construct a vector for coexpressing the antibody H chain and L chain.

[Expression of Recombinant IgG]

32.6 μg of the antibody H chain and L chain coexpression vector prepared by the method described above in [Construction of recombinant IgG expression vector] was diluted with 1.6 mL of opti-MEM (manufactured by Gibco/Thermo Fisher Scientific Inc.) and mixed with 65 μL of Transficient Transfection Reagent (manufactured by Medical & Biological Laboratories Co., Ltd.) diluted with 1.6 mL of opti-MEM, and the mixture was incubated at room temperature for 10 minutes. Then, the mixture was mixed with CHO-K1 cells (1×10⁷ cells) suspended in 10 mL of a DMEM culture solution, followed by culture. 4 hours later, a serum-free medium (Free Style expression CHO media [manufactured by Gibco/Thermo Fisher Scientific Inc.]) was added thereto, and the mixture was further cultured for 4 to 6 days to recover a culture supernatant containing a recombinant antibody.

[Affinity Purification of Antibody]

An empty column (manufactured by Bio-Rad Laboratories, Inc.) was packed with Protein G Sepharose 4 Fast Flow (manufactured by GE Healthcare Japan Corp.) or Bipo Resin Protein L (manufactured by Protein Express) at 1 mL bed volume. Then, the column resin was washed with PBS in an amount of 10 times the bed volume. The culture supernatant filtered through a 0.22 micron filter was added to the column so that the antibody was entrapped to protein G or protein L within the column. Then, the column was washed with PBS in an amount of 10 times the bed volume to wash off nonspecifically adsorbed contaminants. The antibody was eluted using a 100 mM glycine-HCl (pH 2.7) solution, and pH of the eluate was neutralized with 1 M Tris-HCl (pH 8.5). Absorbance at 280 nm was measured with an absorbance meter nanoDrop (manufactured by Thermo Fisher Scientific Inc.), and the antibody concentration was calculated. Expression vectors were also designed and prepared by the same method as above as to the GC33 antibody and the GC199 antibody used as competitive antibodies.

1-2 Results [Antiserum Evaluation of Immunized Mouse]

Blood was collected from SKG/Jcl mice immunized four times with recombinant GPC3, and the production of an antibody against GPC3 in serum was confirmed. As a result, an antibody having binding activity against GPC3 was detected by experiments of ELIS on recombinant GPC3 and FCM on GPC3-expressing cells. Two mice having a particularly high antibody titer (individual Nos. 1413 #2 and 1413 #3) among the mice were used as sources for the preparation of an antibody library.

[Construction of Phage Library]

The number of members in a scFv library estimated by calculation from transformation efficiency was 5.8×10⁷ for mouse 1413 #2 and 4.3×10⁸ for mouse 1413 #3. The immunoglobulin library prepared in this Example was a library prepared from the mice found to produce antibodies in response to the target antigen by immunization with the antigen GPC3. Therefore, a feature of this library is the high possibility of containing the target antibody gene even if the library size is small. Another advantageous feature thereof is that the library contains an antibody that forms a correct conformation in vivo, as compared with a random synthetic antibody library.

[Classification of Clone by Sequence Analysis of Monoclonal scFv]

The DNA sequence analysis of picked up monoclonal scFv was conducted to perform clone classification excluding overlap. As a result, candidate clones were identified as 7 types from D series of the mouse 1413 #2 library, 5 types from E series thereof, 3 types from D series of the mouse 1413 #3 library, and 9 types from E series thereof. The nucleotide sequences of heavy chain and light chain variable regions of these candidate clones were analyzed to exclude overlapping identical clones. As a result, a total of 18 types of scFv clones, i.e., 9 types of scFv clones derived from the mouse 1413 #2 library, and 9 types of scFv clones derived from the mouse 1413 #3 library, were identified.

[Epitope Mapping Analysis of Anti-GPC3 scFv Clone]

18 types of scFv clones identified according to the method described above in the section [Classification of clone by sequence analysis of monoclonal scFv] were used to analyze binding to each GPC3 by FCM using 3 types of cell lines (GPC3 N-terminal fragment-expressing cell line, GPC3 C-terminal fragment-expressing cell line, and GPC3-expressing cell line). As a result, among the 18 types of scFv clones, 14 types (TF1413-02d028, 02d030, 02d039, 02e004, 02e014, 02e030, 02e040, 03e001, 03e004, 03e005, 03e015, 03e019, 03e027, and 03e034) bound to full-length GPC3 and the GPC3 N-terminal fragment (polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 155), but did not bind to the GPC3 C-terminal fragment (polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 156) (see FIG. 2). On the other hand, the existing anti-GPC3 antibodies GC33 (manufactured by Chugai Pharmaceutical Co., Ltd.) and GC199 (manufactured by Chugai Pharmaceutical Co., Ltd.) bound to full-length GPC3 and the GPC3 C-terminal fragment, but did not bind to the GPC3 N-terminal fragment.

From these results, the 14 types of novel scFv clones described above recognizing a GPC3 N-terminal epitope different from a GPC3 C-terminal epitope for the existing anti-GPC3 antibodies (GC33 and GC199) were identified.

Among the 14 types of scFv clones thus identified, top 11 scFv clones (TF1413-02d028, 02d039, 02e004, 02e014, 02e030, 02e040, 03e001, 03e004, 03e005, 03e015, and 03e034) having particularly high binding strength were selected. Table 1 shows the correspondence of SEQ ID NOs to the H chain and L chain V regions of these 11 types of scFv clones. Table 2 shows the correspondence of SEQ ID NOs to the H chain CDR1 to CDR3 of these 11 types of scFv clones. Table 3 shows the correspondence of SEQ ID NOs to the L chain CDR1 to CDR3 of these 11 types of scFv clones.

TABLE 1 scFv clone name and V region SEQ ID NO TF1413-02d028 H chain V region 7 TF1413-02d039 H chain V region 17 TF1413-02e004 H chain V region 27 TF1413-02e014 H chain V region 37 TF1413-02e030 H chain V region 47 TF1413-02e040 H chain V region 57 TF1413-03e001 H chain V region 67 TF1413-03e004 H chain V region 77 TF1413-03e005 H chain V region 87 TF1413-03e015 H chain V region 97 TF1413-03e034 H chain V region 107 TF1413-02d028 L chain V region 8 TF1413-02d039 L chain V region 18 TF1413-02e004 L chain V region 28 TF1413-02e014 L chain V region 38 TF1413-02e030 L chain V region 48 TF1413-02e040 L chain V region 58 TF1413-03e001 L chain V region 68 TF1413-03e004 L chain V region 78 TF1413-03e005 L chain V region 88 TF1413-03e015 L chain V region 98 TF1413-03e034 L chain V region 108

TABLE 2 Clone name and CDR SEQ ID NO TF1413-02d028 H chain CDR1 1 H chain CDR2 2 H chain CDR3 3 TF1413-02d039 H chain CDR1 11 H chain CDR2 12 H chain CDR3 13 TF1413-02e004 H chain CDR1 21 H chain CDR2 22 H chain CDR3 23 TF1413-02e014 H chain CDR1 31 H chain CDR2 32 H chain CDR3 33 TF1413-02e030 H chain CDR1 41 H chain CDR2 42 H chain CDR3 43 TF1413-02e040 H chain CDR1 51 H chain CDR2 52 H chain CDR3 53 TF1413-03e001 H chain CDR1 61 H chain CDR2 62 H chain CDR3 63 TF1413-03e004 H chain CDR1 71 H chain CDR2 72 H chain CDR3 73 TF1413-03e005 H chain CDR1 81 H chain CDR2 82 H chain CDR3 83 TF1413-03e015 H chain CDR1 91 H chain CDR2 92 H chain CDR3 93 TF1413-03e034 H chain CDR1 101 H chain CDR2 102 H chain CDR3 103

TABLE 3 Clone name and CDR SEQ ID NO TF1413-02d028 L chain CDR1 4 L chain CDR2 5 L chain CDR3 6 TF1413-02d039 L chain CDR1 14 L chain CDR2 15 L chain CDR3 16 TF1413-02e004 L chain CDR1 24 L chain CDR2 25 L chain CDR3 26 TF1413-02e014 L chain CDR1 34 L chain CDR2 35 L chain CDR3 36 TF1413-02e030 L chain CDR1 44 L chain CDR2 45 L chain CDR3 46 TF1413-02e040 L chain CDR1 54 L chain CDR2 55 L chain CDR3 56 TF1413-03e001 L chain CDR1 64 L chain CDR2 65 L chain CDR3 66 TF1413-03e004 L chain CDR1 74 L chain CDR2 75 L chain CDR3 76 TF1413-03e005 L chain CDR1 84 L chain CDR2 85 L chain CDR3 86 TF1413-03e015 L chain CDR1 94 L chain CDR2 95 L chain CDR3 96 TF1413-03e034 L chain CDR1 104 L chain CDR2 105 L chain CDR3 106 [Conversion of Anti-GPC3 scFv Antibody to IgG and its Ability to Bind]

The H chain and L chain variable regions of the 11 types of scFv clones selected as described above were bound to mouse IgG constant regions, and full-length recombinant antibodies were expressed using a vector for recombinant IgG expression and affinity-purified. The ability of these IgG antibodies to bind to the GPC3 N-terminal fragment was analyzed using the GPC3 N-terminal fragment-expressing cell line. As a result, 9 types of IgG clones (TF1413-02d028, 02d039, 02e004, 02e014, 02e030, 02e040, 03e004, 03e005, and 03e034) maintained binding activity against the GPC3 N-terminal fragment, whereas the remaining two types of IgG clones (TF1413-03e001 and 03e015) lacked binding activity against the GPC3 N-terminal fragment (see FIG. 3). The 9 types of IgG clones described above did not bind to the GPC3 C-terminal fragment (see FIG. 3).

These results indicate that among the 11 types of scFv clones, 9 types (TF1413-02d028, 02d039, 02e004, 02e014, 02e030, 02e040, 03e004, 03e005, and 03e034) are convertible to IgG type. Table 4 shows the correspondence of SEQ ID NOs to the H chains and the L chains of the 11 types of IgG clones.

TABLE 4 IgG clone name and region SEQ ID NO TF1413-02d028 H chain 9 TF1413-02d039 H chain 19 TF1413-02e004 H chain 29 TF1413-02e014 H chain 39 TF1413-02e030 H chain 49 TF1413-02e040 H chain 59 TF1413-03e001 H chain 69 TF1413-03e004 H chain 79 TF1413-03e005 H chain 89 TF1413-03e015 H chain 99 TF1413-03e034 H chain 109 TF1413-02d028 L chain 10 TF1413-02d039 L chain 20 TF1413-02e004 L chain 30 TF1413-02e014 L chain 40 TF1413-02e030 L chain 50 TF1413-02e040 L chain 60 TF1413-03e001 L chain 70 TF1413-03e004 L chain 80 TF1413-03e005 L chain 90 TF1413-03e015 L chain 100 TF1413-03e034 L chain 110

Example 2

2. Binding Activity of Novel Anti-GPC3 Antibody Against GPC3 Treated with EDTA (Ethylenediaminetetraacetic Acid), Trypsin or Collagenase [Preparation of Cell Treated with EDTA or Trypsin]

A SK-Hep-1 cell line forced to express GPC3 was cultured in two T-75 flasks. The culture supernatant of each flask was aspirated, and the flask was washed with 3 mL of PBS. Then, 3 mL of 0.02% EDTA/PBS solution (hereinafter, simply referred to as “EDTA”) or 0.05% trypsin solution (hereinafter, simply referred to as “trypsin”) was added to each flask. Each flask was incubated at 37° C. for 5 minutes (EDTA) or 2 minutes and 30 seconds (trypsin) to dissociate the cells from the flask. Then, 7 mL of a DMEM culture solution was added to each flask. After pipetting, the cell suspension was recovered into each 50 mL conical tube. Each flask was further washed with 10 mL of a DMEM culture solution. Then, the recovered washes were also recovered into the 50 mL conical tube containing each cell suspension, followed by centrifugation (1,500 rpm, 4° C., 4 min). After aspiration of the supernatant from each conical tube, 10 mL of a DMEM culture solution was added to the pellet, and the number of cells dissociated with EDTA or trypsin was counted.

The cells treated with EDTA or trypsin were adjusted to 2×10³ cells/tube and subjected to FACS (EC800) analysis. The FACS analysis employed 3 types of antibodies (fluorescently APC-labeled anti-mouse IgG antibody [5 μg/tube; manufactured by BioLegend, Inc.], GC33 antibody [1.0 μg/tube; manufactured by Medical & Biological Laboratories Co., Ltd. Life Science], and scFv clone [TF1413-02d028] antibody described above [1.0 μg/tube]).

[Preparation of Cell Treated with Collagenase]

1×10⁶ cells dissociated with EDTA as described above were placed in a 50 mL conical tube and centrifuged (1,500 rpm, 4° C., 4 min), and the supernatant was aspirated to prepare a cell mass (pellet). 5 mL of a collagenase P solution was added to the pellet, and the mixture was incubated at 37° C. for 30 minutes to prepare a cell suspension. Then, the cell suspension was passed through a 100 μm cell strainer while washed with 30 mL of a DMEM culture solution. The cell suspension was passed again through a 100 μm cell strainer and centrifuged (300 g, 4° C., 10 min), and the supernatant was aspirated. The pellet was washed by the addition of 20 mL of PBS and then centrifuged (300 g, 4° C., 5 min), and the supernatant was aspirated. The cells were suspended by the addition of 5 mL of a DMEM culture solution. Then, the number of cells was counted, and 2×10³ cells/tube were analyzed by FACS (EC800). The FACS analysis employed 3 types of antibodies (fluorescently APC-labeled anti-mouse IgG antibody [5 μg/tube; manufactured by BioLegend, Inc.], GC33 antibody [1.0 μg/tube; manufactured by Medical & Biological Laboratories Co., Ltd. Life Science], and scFv clone [TF1413-02d028] antibody described above [1.0 μg/tube]), as in the cells treated with EDTA or trypsin. The results are shown in FIG. 4. In FIG. 4, the right peak on the abscissa represents that the GC33 antibody or the scFv clone [TF1413-02d028] antibody bound to the GPC3 protein.

[Results]

As shown in FIG. 4, the binding activity of the antibody of the present invention (TF1413-02d028) against the GPC3 protein treated with trypsin or collagenase was markedly decreased. These results indicate that the antibody of the present invention specifically recognizes the conformation of the GPC3 protein, suggesting that the antibody of the present invention has high specificity in vivo.

Example 3 3. Development of GPC3 CAR-T Cell Using Novel Anti-GPC3 Antibody [Summary]

GPC3 is a cell surface molecule, the expression of which is not observed in human adult tissues except for placenta, but is observed in tissues of various cancers such as hepatocellular carcinoma, melanoma, ovarian clear cell adenocarcinoma, and lung squamous cell carcinoma. Thus, GPC3 is capable of serving as a target molecule in CAR-T cell therapy exploiting a chimeric antigen receptor (CAR). Accordingly, GPC3 CAR-T cells were prepared using 11 types of scFv clones prepared in Example 1 and analyzed for cancer cytotoxic activity and the ability to produce interferon γ (IFN-γ).

[Preparation of GPC3 CAR Vector]

scFv having a V_(H)-linker-V_(L) sequence was designed as to 11 types of scFv clones (TF1413-02d028, 02d039, 02e004, 02e014, 02e030, 02e040, 03e001, 03e004, 03e005, 03e015, and 03e034) prepared in Example 1, on the basis of their respective amino acid sequences of V_(H) and V_(L) (see Table 5). The linker used consisted of 15 amino acid residues with 3 repeats of a polypeptide “GGGGS”. A human immunoglobulin H chain-derived signal sequence consisting of the amino acid sequence represented by SEQ ID NO: 188 was added to the N terminus of V_(H).

TABLE 5 SEQ ID NO: 165: TF1413-02d028 -derivedscFv QVQLKESGPELEKPGASVKISCKASGYSFTGYNMNWVKQSNGKSLEWI GNIDPYYGGTSYNQKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYC

ARGDYRAYYFDYWGQGTTLTVSGGGGSGGGGSGGGGSDIQMTQSPKFM STSVGDRVSITCKASQNVRTAVAWYQQKPGQSPKALIYLASNRHTGVP DRFTGSGSGTDFTLTISNVQSEDLADYFCLQHWNYPLTFGAGTKLELK R SEQ ID NO: 166: TF1413-02d039 -derived scFv EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWV AYISSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYC

SLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGAGTKL ELKR SEQ ID NO: 167: TF1413-02e004 -derived scFv QVQLQQSGAELVKPGAPVKLSCKASGYTFTSYWMNWVKQRPGRGLEWI GRIDPSDSETHYNQKFKDEATLTVDKSSSTAYIQLSSLTSEDSAVYYC

VGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRF TGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPTFGGGTKLEIKR SEQ ID NO: 168: TF1413-02e014 -derived scFv QVQLKQSGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEQGLEWI GWIDPENGDTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYC

FMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTG VPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPLTFGGGTKLE IKR SEQ ID NO: 169: TF1413-02e030 -derived scFv EVQLQQSGAELVRPGALVKLSCKASGFNIKDYYMHWVKQRPEQGLEWI GWIDPENGNTIYDPKFQGKASITADTSSNTAYLQLSSLTSEDTAVYYC

MSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTR ESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPLTFGAGT KLELKR SEQ ID NO: 170: TF1413-02e040 -derived scFv EVMLVESGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWI GLINPYNGGTSYNQNFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYC

SVGDRVSITCKASQNVRTAVAWYQQKPGQSPKALIYLASNRHTGVPDR FTGSGSGTDFTLTISNVQSEDLADYFCLQHWNYPLTFGAGTKLELKR SEQ ID NO: 171: TF1413-03e001 -derived scFv QVQLKQSGPELVKPGASVKISCKASGYSFTGYYMHWVKQSHVKSLEWI GRINPYNGATSYNQNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYC

VGDRVSVTCEASQNVDNNVVWYQQKPGQSPKALIYSASYRYSGVPDRF TGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPLTFGAGTKLEIKR SEQ ID NO: 172: TF1413-03e004 -derived scFv QVQLKQSGAELVKPGAPVKLSCKASGYTFTSYWMNWVKQRPGRGLEWI GRIDPSDSETHYNQKFKDKATLTVDKSSSTAYIQLSSLTSEDSAVYYC

TSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPD RFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPLTFGAGTKLELKR SEQ ID NO: 173: TF1413-03e005 -derived scFv QVQLKESGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEQGLEWI DPENGDTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNAF

ASLGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASTLDSGVPK RFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPLTFGAGTKLELKR SEQ ID NO: 174: TF1413-03e015 -derived scFv EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWI GLINPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYC

MSTSGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSASYRYSGVP DRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNRYPLTFGVGTKLEIK R SEQ ID NO: 175: TF1413-03e034 -derived scFv EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQSNGKSLEWI GNIDPYYGGTSYNQKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYC

STSVGDRVSITCKASQNVRTAVAWYQQKPGQSPKALIYLASNRHTGVP DRFTGSGSGTDFTLTISNVQSEDLADYFCLQHWNYPLTFGAGTKLELK R

In the tables, the linker is boxed in a double line, V_(H) is underlined with a single line, and V_(L) is underlined with a double line.

A nucleotide sequence encoding each anti-GPC3 scFv of Table 5 was synthesized by optimization for human codons and inserted to a CAR expression vector. The CAR gene used had a gene encoding a fusion peptide (peptide consisting of the amino acid sequence represented by SEQ ID NO: 185) consisting of a human CD8-derived transmembrane region and a human CD28/4-1BB/CD3 zeta-derived immunocompetent cell activation signal transduction region, a 2A self-cleaving sequence, human IL-7 gene, a 2A self-cleaving sequence, human CCL19 gene, a 2A self-cleaving sequence, and HSV-TK gene, downstream of the scFv gene, and the whole was incorporated into a MSGV1 retrovirus vector (see International Publication No. WO 2016/056228).

[Preparation of GPC3 CAR-T Cell]

The GPC3 CAR vectors derived from the 11 types of scFv clones described above were each transiently introduced to GP2 packaging cells to prepare retrovirus vectors. T cells were infected with these vectors for gene transfer to induce GPC3 CAR-T cells. The ratio of GPC3 CAR-expressing cells to the gene-transferred T cells varied from 5.3 to 39.2%. Accordingly, the following function assay was carried out using GPC3 CAR-T cells derived from 5 types of scFv clones (TF1413-02d028, TF1413-02d039, TF1413-02e014, TF1413-02e030, and TF1413-03e005) that exhibited 25% or more of the ratio.

[Damaging Activity of GPC3 CAR-T Cell Against GPC3-Expressing Cell Line]

In order to study the damaging activity of the GPC3 CAR-T cells against cancer cells, coculture assay was carried out using the GPC3 CAR-T cells and a GPC3-expressing cell line, i.e., a hepatocellular carcinoma-derived cell line Sk-HEP-1 caused to express GPC3 (Sk-HEP-1 GPC3 cell line), or a cell line expressing no GPC3 (Sk-HEP-1 mock cell line). The GPC3 CAR-T cells were mixed with the target cancer cells (Sk-HEP-1 GPC3 cell line or Sk-HEP-1 mock cell line) at a ratio of 1:1 (1×10³ cells/well) and cultured in a 24-well plate. 48 hours later, the cells were recovered, stained with an anti-CD45 antibody, and analyzed by FCM with CD45-positive cells as GPC3 CAR-T cells and CD45-negative cells as residual cancer cells [Sk-HEP-1 GPC3 cells]. As a result, all the GPC3 CAR-T cells derived from the 5 types of scFv clones described above almost completely damaged the Sk-HEP-1 GPC3 cells, but did not exhibit damaging activity against the Sk-HEP-1 mock cells (see FIGS. 5 and 6). In the case of using cells uninfected with the virus vector (non-gene-transferred cells [“Non infection” in FIGS. 5 and 6]) as a negative control for the GPC3 CAR-T cells, these cells exhibited damaging activity neither against the Sk-HEP-1 GPC3 cells nor against the Sk-HEP-1 mock cells.

From these results, the GPC3CAR-T cells derived from the selected 5 types of anti-GPC3 scFv clones (TF1413-02d028, TF1413-02d039, TF1413-02e014, TF1413-02e030, and TF1413-03e005) were shown to specifically exert cytotoxic activity against cancer cells expressing GPC3.

[Ability of GPC3 CAR-T Cell to Produce IFN-γ by Recognizing GPC3-Expressing Cell]

In addition to the damaging activity against GPC3-expressing (positive) cancer cells, the ability of the GPC3 CAR-T cells to produce IFN-γ was analyzed. The GPC3 CAR-T cells were mixed with the target cancer cells (Sk-HEP-1 GPC3 cell line or Sk-HEP-1 mock cell line) at a ratio of 1:1 (1×10³ cells/well) and cultured for 48 hours in a 24-well plate, and the concentration of IFN-γ produced into the culture supernatant was measured by ELISA. As a result, all the GPC3 CAR-T cells derived from the 5 types of scFv clones described above exhibited the ability to produce IFN-γ in a manner dependent on the expression of GPC3. Particularly, the GPC3 CAR-T cells derived from clone TF1413-02d028 exhibited the highest ability to produce IFN-γ (see FIG. 7).

Example 4 4. Preparation of Humanized Antibody

scFv humanized antibodies were designed on the basis of two types of scFv clones (TF1413-02d028 and 02d039) prepared in Example 1 (see Table 6). The linker used consisted of 15 amino acid residues with 3 repeats of a polypeptide “GGGGS”. A human immunoglobulin H chain-derived signal sequence consisting of the amino acid sequence represented by SEQ ID NO: 188 was added to the N terminus of V_(H).

TABLE 6 SEQ ID NO: 178: #5 VH1-15-VL1 (TF1413- 02d028-derived scFv humanized antibody 1) QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYNMNWVRQAPGQGLEWI GNIDPYYGGTSYNQKFKGRATLTVDTSTSTAYMELRSLRSDDTAVYYC

LSASVGDRVTITCKASQNVRTAVAWYQQKPGKAPKALIYLASNRHTGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHWNYPLTFGGGTKVEI K SEQ ID NO: 179: #5 VH2-15-VL1 (TF1413-  02d028-derived scFv humanized antibody 2) QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYNMNWVRQAPGQGLEWI GNIDPYYGGTSYNQKFKGRVTLTVDTSTSTAYMELRSLRSDDTAVYYC

LSASVGDRVTITCKASQNVRTAVAWYQQKPGKAPKALIYLASNRHTGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHWNYPLTFGGGTKVEI K SEQ ID NO: 180: #5 VH3-15-VL1 (TF1413- 02d028-derived scFv humanized antibody 3) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYNMNWVRQAPGQGLEWI GNIDPYYGGTSYNQKFKGRVTLTVDTSTSTAYMELRSLRSDDTAVYYC

LSASVGDRVTITCKASQNVRTAVAWYQQKPGKAPKALIYLASNRHTGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHWNYPLTFGGGTKVEI K SEQ ID NO: 181: #6 VH1-15-VL1 (TF1413- 02d039-derived scFv humanized antibody 1) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLEWV AYISSGGGSTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC

ARRGLRRAMDYWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSL PVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGT KVEIK SEQ ID NO: 182: #6 VH1-15-VL2 (TF1413- 02d039-derived scFv humanized antibody 2) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLEWV AYISSGGGSTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC

PVTPGEPASISCRSSQSLVHSSGNTYLHWYLQKPGQSPQLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGT KVEIK SEQ ID NO: 183: #6 VH2-15-VL1 (TF1413- 02d039-derived scFv humanized antibody 3) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKRLEWV AYISSGGGSTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC

PVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGT KVEIK SEQ ID NO: 184: #6 VH2-15-VL2 (TF1413- 02d039-derived scFv humanized antibody 4) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKRLEWV AYISSGGGSTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC

PVTPGEPASISCRSSQSLVHSSGNTYLHWYLQKPGQSPQLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGGGT KVEIK

In the tables, the linker is boxed in a double line, V_(H) is underlined with a single line, and V_(L) is underlined with a double line.

INDUSTRIAL APPLICABILITY

The present invention contributes to the field of cancer immunotherapy. 

1. A single chain antibody specifically binding to a human GPC3 (glypican-3)-derived polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 155, wherein the single chain antibody (1-1) comprises a heavy chain complementarity determining region (CDR) 1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3, and a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:
 6. 2. The single chain antibody according to claim 1, wherein the single chain antibody (1-2) comprises a heavy chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 7, and a light chain variable region consisting of an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO:
 8. 3. The single chain antibody according to claim 1, wherein the single chain antibody (1-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO:
 165. 4. The single chain antibody according to claim 1, wherein the single chain antibody (1-3′-1) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 178; or (1-3′-2) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO: 179; or (1-3′-3) comprises an amino acid sequence having at least 80% or higher sequence identity to the amino acid sequence represented by SEQ ID NO:
 180. 5. A chimeric antigen receptor (CAR) comprising the single chain antibody according to claim 1, a transmembrane region fused with a carboxyl terminus of the single chain antibody, and an immunocompetent cell activation signal transduction region fused with a carboxyl terminus of the transmembrane region.
 6. The CAR according to claim 5, comprising the amino acid sequence represented by any of SEQ ID NOs: 185 to
 187. 7. An immunocompetent cell expressing the CAR according to claim
 5. 8. The immunocompetent cell according to claim 7, further expressing interleukin 7 (IL-7) and chemokine ligand 19 (CCL19).
 9. A nucleotide sencoding the single chain antibody according to claim
 1. 10. A nucleotide encoding the CAR according to claim
 5. 11. A vector comprising a promoter, and the nucleotide according to claim 9 operably linked downstream of the promoter.
 12. A vector comprising a promoter, and the nucleotide encoding the CAR according to claim 10 operably linked downstream of the promoter.
 13. A host cell in which the vector according to claim 11 has been introduced.
 14. A host cell in which the vector according to claim 12 has been introduced. 